Attorney's Docket No.: 06618-343002 / CIT 2857D
APPLICATION
FOR
UNITED STATES LETTERS PATENT
TITLE: METHUSELAH GENE, COMPOSITIONS AND METHODS
OF USE
APPLICANT: YI-JYUN LIN AND SEYMOUR BENZER
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Attorney Docket No. 06618/343002
METHUSELAH GENE, COMPOSITIONS AND METHODS OF USE
The U.S. Government has certain rights in this invention
pursuant to Grant Nos . EY09278 and AG12289 awarded by the
National Institute of Health, and Grant No. MCB9408718 awarded
by the National Science Foundation.
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from Provisional Application
Serial No. 60/095,826, filed August 7, 1998, which is
incorporated herein by reference in its entirety and to which
application a priority claim is made under 35 U.S.C. §119 (e) .
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides,
polypeptides encoded by such polynucleotides, the use of such
polynucleotides and polypeptides, as well as the production and
isolation of such polynucleotides and polypeptides. More
particularly, the polynucleotides and polypeptides of the
present invention have been identified as a G-protein-coupled
receptors having stress associated and life span associated
activities .
BACKGROUND OF THE INVENTION
Studies on the genetics of aging in a number of organisms
including the yeast Saccharomyces cerevisiae, the roundworm
Caenorhabditis elegans, and the fruit fly Drosophila
melanogaster have revealed the role of metabolic capacity and
resistance to stress in determining life span. One mode of
modulation of longevity has been suggested to be signal
transduction. Signal transduction has emerged as an important
molecular mechanism underlying longevity. The results obtained
from the study of these organisms are applicable to the dietary
Attorney Docket No. 06618/343 002
restriction paradigm in mammals. It is thought that many of the
molecular characteristics identified from these studies will be
of interest in determining the effect of diet and signal
transduction in the life span of mammals. However, the
identification and role of the genes and gene products
responsible for modulating the life-span of organism are not yet
fully understood. Accordingly, there is a desire to obtain and
characterize life-span modulating genes in order to more fully
understand the role of stress and life-span.
The effect of genes on life span in Drosophila has been
established by selective breeding (Rose et al . Genetics, 97,
173-186 (1981)). However, that methodology involves the
participation of multiple genes with additive and quantitative
effects that can be difficult to unravel. A more incisive
approach is to use single gene mutations. A search for life-
extension mutants can lead to the identification of individual
genes that regulate biological aging.
SUMMARY OF THE INVENTION
In a first embodiment, the present invention provides a
substantially purified Methuselah (MTH) polypeptide having an
amino acid sequence as set forth in SEQ ID NO: 2.
In another embodiment, the present invention provides an
isolated polynucleotide encoding an amino acid sequence as set
forth in SEQ ID N0:2. The isolated polynucleotide is selected
from the group consisting of SEQ ID NO;l; SEQ ID N0:1, wherein T
can also be U; a nucleic acid sequence complementary to SEQ ID
N0:1; and fragments of a), b) , or c) that are at least 15 bases
in length and that hybridize under stringent conditions to DNA
which encodes the polypeptide of SEQ ID NO: 2.
In another embodiment, the present invention provides an
expression vector containing an mth polynucleotide. The vector
can be for example, a plasmid or a viral vector.
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In yet another embodiment, the present invention provides a host
cell transformed with an expression vector containing an mth
polynucleotide .
In yet a further embodiment, the present invention provides a
method of producing an MTH polypeptide by transforming a host
cell with an mth polynucleotide; expressing the polynucleotide
in the host; and recovering the MTH polypeptide.
In another embodiment, an antibody that binds to the polypeptide
of SEQ ID NO: 2 is provided. The antibody can be polyclonal or
monoclonal .
The present invention also provides a method for identifying a
compound which modulates mth expression or activity comprising:
incubating components comprising the compound and an MTH
polypeptide, or a recombinant cell expressing an MTH
polypeptide, under conditions sufficient to allow the components
to interact; and determining the effect of the compound on the
expression or activity of the gene or polypeptide, respectively.
In yet another embodiment, the present invention provides a
method of detecting an mth- specific cell component in a sample
comprising: contacting a sample suspected of containing mth with
a reagent that binds to the mth- specific component; and
detecting binding of the reagent to the component.
In yet a further embodiment, the present invention provides a
method of promoting insect cell survival in vitro comprising
contacting the cell with a survival promoting amount of a
compound containing an MTH polypeptide or a agent capable of
modulating MTH activity or expression.
In yet another embodiment, the present invention provides a
method of producing a non-human organism having an increased
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life span comprising: introducing a transgene disrupting or
interfering with expression of Methuselah (mth) into germ cells
of a pronuclear embryo of the organism; implanting the embryo
into the oviduct of a pseudopregnant female thereby allowing the
embryo to mature to full term progeny; testing the progeny for
presence of the transgene to identify transgene-positive
progeny; and cross-breeding transgene-positive progeny to obtain
further transgene-positive progeny.
In yet another embodiment, the present invention provides a
transgenic organims having a phenotype characterized by an
increase in mass or an increase in life span or an increase in
resistance to a biologic stress. The organism may be any non-
human organsims, including, for example, bovine, porcine and
invetebrates , such as Drosophila.
In another embodiment, the present invention provides a method
of increasing the life span of a subject, comprising:
administering to the subject, a reagent which affects mth
activity or expression.
In yet a further embodiment, the present invention provides a
kit useful for the detection of an MTH polypeptide, the kit
comprising a carrier containing one or more containers
comprising a first container containing a mth binding reagent.
These and other aspects of the present invention will be
apparent to those of skill in the art from the teachings herein.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings and figures are illustrative of
embodiments of the invention and are not meant to limit the
scope of the invention as encompassed by the claims.
FIG. 1 shows the life span extension effects of Methuselah.
Male flies of the parental strain (white 111 *) and Methuselah
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(homozygous for the P-element insertion) were maintained in a
constant temperature, humidity, and 12/12 -hour dark/light cycle
environment. Flies were transferred to fresh food vials
containing standard cornmeal agar medium and scored for survival
every three to four days. The average life span for w 1118 and mth
were 57 and 77 days, respectively. The numbers of flies tested
were 876 for w 1118 and 783 for mth.
FIG. 2 shows the stress responses in Methuselah flies. Flies
homozygous for the mth mutation were compared with those
containing the corresponding wild-type allele. Newly eclosed
flies were sex-segregated, distributed 20 per vial, and
maintained in fresh cornmeal food vials for 2-5 days before
y testing. Genotypes and sexes are indicated. A. Paraquat
;yD resistance. Flies (age two days) were starved for 6 hours, then
transferred to vials (2.5 cm x 9.3 cm) containing two 2.4 cm
J: glass fiber filter circles (Whatman) wetted with 20 mM paraquat
(Sigma) in 5% sucrose solution, and survival scored at 25 °C. B.
a Starvation test. Flies (age two days) were transferred to vials
% containing filters moisturized with 0.2 ml of distilled water.
Distilled water was added to keep the filters moist during the
pi test. C. Thermal stress test. Flies (age five days) were
transferred to vials containing 1% agar in 5% sucrose solution,
and maintained at 36°C. Initially, immobilized flies were scored
every 3 0 minutes; as immobilization accelerated, scoring was
done every 5 minutes.
FIG. 3 shows the mth gene. The full-length cDNA and its
corresponding genomic segment are shown. A. The genomic DNA.
The letters represent restriction enzyme sites; E, EcoR I; P,
Pst I; Sa, Sac I, Sm, Sma I, X, Xba I. Boxes indicate exons;
hatched boxes the open reading frame. The P-element insertion
site is indicated by an arrow. The two plasmid rescue clones,
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44P1 and 44E1 represent, respectively, upstream and downstream
fragments relative to the P-element. The structure is based on
the genomic sequence derived from the PI plasmid, DS06S92 of the
BDGP . B. cDNA and protein sequence. Nucleotides are in plain
letters, amino acids in italics; numbers of the nucleotide and
amino acid sequence are indicated to the left and right,
respectively. The putative leader peptide sequence is in bold
face; transmembrane domains are underlined. The polyadenylation
site is boxed. The sequence is derived from LD08316 of the
BDGP. C. Hydropathic profile of the MTH protein, analyzed by
the Kyte-Doolittle algorithm. The seven hydrophobic regions
(excluding the N-terminal putative leader peptide) are
designated.
FIG. 4 shows the alignment of MTH with several known G-protein
coupled receptors. The predicted MTH protein is aligned to
partial sequences of the human leukocyte surface antigen CD97
(hCD97, GenBank accession number P48960) , rat a-latrotoxin
receptor (rLR, U72487) , and mouse EGF-module- containing receptor
(mEMR-l, Q61549) . Dark shading indicates identity, gray shading
similarity. The seven transmembrane domains of MTH are
indicated by lines above each row. Consensus amino acids are
cited below; similar residues are indicated by dots.
FIG. 5 shows the expression in wild type (e.g., w 1118 ) flies
compared to mth mutant flies (P+/mth+) in the head region of
Drosophila flies. The expression of mth was reduced to about
10%.
FIG. G shows the expression of mth in wild type flies compared
to mth flies (P+/mth+) in the thoracic region of the fly.
FIG. 7 shows a gel from an RNAse protection assay comparing the
expression of mth in wildtype flies and mth mutant flies
(P+/mth+) . Lane 5 represents RNA from mth adult flies,
demonstrating a reduction in expression of mth in P+/mth+
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Drosophila compared to lane 6. Lane 6 shows RNA from wildtype
Drosophila (mth+/mth+) . Lane 7 and 8 demonstrate the difference
in expression of mth in mutant flies (lane 7) compared to
wildtype (lane 8) in Drosophila embryos.
FIG. 8 shows the localization and expression of mth in
Drosophila mth mutants and wildtype flies using a monoclonal
antibody to mth and developed with an anti-mouse secondary
antibody and FITC. The left series of panels represents
wildtype flies (i.e., panels A, C, E, and G) and the right
series of panels represents the mth mutant flies (i.e., panels
B 7 D, F, and H) . Panels A-B are from the trunk thoracic muscles
of the flies. Panels C-D are from the ventral layer of the
thoracic region. Panels E-F are from leg muscles of the flies.
Panels G-H are from the proboscis muscles of the flies.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides polypeptides and polynucleotides
encoding the polypeptides, wherein each polypeptide is
characterized as a stress related or life span modulating
polypeptide termed, herein, a Methuselah polypeptide.
The present invention originated from the discovery and cloning
of a stress related gene termed Methuselah (mth) , which encode a
polypeptide (MTH) identified from invertebrates (e.g.,
Drosophila) . This gene, referred to as mth, encodes a
polypeptide which affects life span and susceptibility to
biological stress factors. The demonstration of life span
enhancing and stress resistance activity of the Drosophila mth
family member raises the possibility that a mammalian family
member may have similar functions, and that altering the
activity (i.e., enhancing or reducing) may be important in
promoting the life span of cells and subjects as well as
promoting resistance to biological stress.
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To facilitate understanding of the invention, a number of terms
are defined below.
The term "isolated" means altered "by the hand of man" from its
natural state; i.e., if it occurs in nature, it has been changed
or removed from its original environment, or both. For example,
a naturally occurring polynucleotide or a polypeptide naturally
present in a living animal in its natural state is not
"isolated", but the same polynucleotide or polypeptide separated
from the coexisting materials of its natural state is
"isolated", as the term is employed herein.
As part of or following isolation, a polynucleotide can be
joined to other polynucleotides, such as for example DNAs, for
mutagenesis studies, to form fusion proteins, and for
propagation or expression of the polynucleotide in a host. The
isolated polynucleotides, alone or joined to other
polynucleotides, such as vectors, can be introduced into host
cells, in culture or in whole organisms. Such polynucleotides,,
when introduced into host cells in culture or in whole
organisms, still would be isolated, as the term is used herein,
because they would not be in their naturally occurring form or
environment. Similarly, the polynucleotides and polypeptides
may occur in a composition, such as a media formulation
(solutions for introduction of polynucleotides or polypeptides,
for example, into cells or compositions or solutions for
chemical or enzymatic reactions which are not naturally
occurring compositions) and, therein remain isolated
polynucleotides or polypeptides within the meaning of that term
as it is employed herein.
The term "ligation" refers to the process of forming
phosphodiester bonds between two or more polynucleotides, which
most often are double stranded DNAs. Techniques for ligation
are well known to the art and protocols for ligation are
described in standard laboratory manuals and references, such
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as, for instance, Sambrook et al . , MOLECULAR CLONING, A
LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York (1989) .
The term "oligonucleotide" as used herein is defined as a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides, preferably more than three, and usually more
than ten. The exact size of an oligonucleotide will depend on
many factors, including the ultimate function or use of the
oligonucleotide. Oligonucleotides can be prepared by any
suitable method, including, for example, cloning and restriction
of appropriate sequences and direct chemical synthesis by a
method such as the phosphotriester method of Narang et al . ,
1979, Meth. Enzymol., 68:90-99; the phosphodiester method of
Brown et al., ±919, Method Enzymol., 68:109-151, the
diethylphosphoramidite method of Beaucage et al . , 1981,
Tetrahedron Lett., 22:1859-1862; the triester method of
Matteucci et al . , 1981, J. Am. Chem. Soc . , 103:3185-3191, or
automated synthesis methods; and the solid support method of
U.S. Patent No. 4,458,066.
The term "plasmids" generally is designated herein by a lower
case p preceded and/or followed by capital letters and/or
numbers, in accordance with standard naming conventions that are
familiar to those of skill in the art.
Plasmids disclosed herein are either commercially available,
publicly available on an unrestricted basis, or can be
constructed from available plasmids by routine application of
well known, published procedures. Many plasmids and other
cloning and expression vectors that can be used in accordance
with the present invention are well known and readily available
to those of skill in the art. Moreover, those of skill readily
may construct any number of other plasmids suitable for use in
the invention. The properties, construction and use of such
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plasmids, as well as other vectors, in the present invention
will be readily apparent to those of skill from the present
disclosure .
"Polynucleotide" or "nucleic acid sequence" refers to a
polymeric form of nucleotides at least 10 bases in length. By
"isolated nucleic acid sequence" is meant a polynucleotide that
is not immediately contiguous with either of the coding
sequences with which it is immediately contiguous (one on the 5 '
end and one on the 3 1 end) in the naturally occurring genome of
the organism from which it is derived. The term therefore
includes, for example, a recombinant DNA which is incorporated
into a vector; into an autonomously replicating plasmid or
virus; or into the genomic DNA of a prokaryote or eukaryote, or
which exists as a separate molecule (e.g., a cDNA) independent
of other sequences. The nucleotides of the invention can be
ribonucleotides, deoxyribonucleotides, or modified forms of
either nucleotide. The term includes single and double stranded
forms of DNA.
The term polynucleotide (s) generally refers to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. Thus, for
instance, polynucleotides as used herein refers to, among
others, single -and double -stranded DNA, DNA that is a mixture of
single- and double -stranded regions, single- and double -stranded
RNA, and RNA that is mixture of single- and double -stranded
regions, hybrid molecules comprising DNA and RNA that may be
single -stranded or, more typically, double -stranded or a mixture
of single- and double -stranded regions.
In addition, polynucleotide as used herein refers to triple-
stranded regions comprising RNA or DNA or both RNA and DNA. The
strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more
of the molecules, but more typically involve only a region of
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some of the molecules. One of the molecules of a triple-helical
region often is an oligonucleotide.
As used herein, the term polynucleotide includes DNAs or RNAs as
described above that contain one or more modified bases. Thus,
DNAs or RNAs with backbones modified for stability or for other
reasons are "polynucleotides" as that term is intended herein.
Moreover, DNAs or RNAs comprising unusual bases, such as
inosine, or modified bases, such as tritylated bases, to name
just two examples, are polynucleotides as the term is used
herein.
It will be appreciated that a great variety of modifications
have been made to DNA and RNA that serve many useful purposes
known to those of skill in the art. The term polynucleotide as
it is employed herein embraces such chemically, enzymatically or
metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of viruses and
cells, including simple and complex cells, inter alia.
Nucleic acid sequences which encode a fusion protein of the
invention can be operatively linked to expression control
sequences. "Operatively linked" refers to a juxtaposition
wherein the components so described are in a relationship
permitting them to function in their intended manner. An
expression control sequence operatively linked to a coding
sequence is ligated such that expression of the coding sequence
is achieved under conditions compatible with the expression
control sequences. As used herein, the term "expression control
sequences" refers to nucleic acid sequences that regulate the
expression of a nucleic acid sequence to which it is operatively
linked. Expression control sequences are operatively linked to
a nucleic acid sequence when the expression control sequences
control and regulate the transcription and, as appropriate,
translation of the nucleic acid sequence. Thus, expression
control sequences can include appropriate promoters, enhancers,
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transcription terminators, a start codon (i.e., ATG) in front of
a protein-encoding gene, splicing signals for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of the mRNA, and stop codons . The term
"control sequences" is intended to include, at a minimum,
components whose presence can influence expression, and can also
include additional components whose presence is advantageous,
for example, leader sequences and fusion partner sequences.
Expression control sequences can include a promoter.
By "promoter" is meant minimal sequence sufficient to direct
transcription. Also included in the invention are those
promoter elements which are sufficient to render promoter-
dependent gene expression controllable for cell- type specific,
tissue-specific, or inducible by external signals or agents ;
such elements may be located in the 5' or 3' regions of the
gene. Both constitutive and inducible promoters, are included
in the invention (see e.g., Bitter et al . , Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of
bacteriophage y, plac, ptrp, ptac (ptrp-lac hybrid promoter) and
the like may be used. When cloning in mammalian cell systems,
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
retrovirus long terminal repeat; the adenovirus late promoter ;
the vaccinia virus 7.5K promoter) may be used. Promoters
produced by recombinant DNA or synthetic techniques may also be
used to provide for transcription of the nucleic acid sequences
of the invention.
In the present invention, the nucleic acid sequences encoding a
fusion protein of the invention may be inserted into a
recombinant expression vector. The term "recombinant expression
vector" refers to a plasmid, virus or other vehicle known in the
art that has been manipulated by insertion or incorporation of
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the nucleic acid sequences encoding the fusion peptides of the
invention. The expression vector typically contains an origin
of replication, a promoter, as well as specific genes which
allow phenotypic selection of the transformed cells. Vectors
suitable for use in the present invention include, but are not
limited to the T7 -based expression vector for expression in
bacteria (Rosenberg, et al., Gene 56:125, 1987), the pMSXND
expression vector for expression in mammalian cells (Lee and
Nathans, J. Biol. Chem. 263:3521, 1988), baculovirus -derived
vectors for expression in insect cells, cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV. The nucleic acid
sequences encoding a fusion polypeptide of the invention can
also include a localization sequence to direct the indicator to
particular cellular sites by fusion to appropriate organellar
targeting signals or localized host proteins. A polynucleotide
encoding a localization sequence, or signal sequence, can be
used as a repressor and thus can be ligated or fused at the 5 1
terminus of a polynucleotide encoding the reporter polypeptide
such that the signal peptide is located at the amino terminal
end of the resulting fusion polynucleotide/polypeptide. The
construction of expression vectors and the expression of genes
in transfected cells involves the use of molecular cloning
techniques also well known in the art. Sambrook et al . ,
Molecular Cloning --A Laboratory Manual , Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols
in Molecular Biology , M. Ausubel et al . , eds . , (Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., most recent Supplement).
These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. (See, for example, the techniques described in
Maniatis, et al . , Molecular Cloning A Laboratory Manual , Cold
Spring Harbor Laboratory, N.Y., 1989).
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Depending on the vector utilized, any of a number of suitable
transcription and translation elements, including constitutive
and inducible promoters, transcription enhancer elements,
transcription terminators, etc. may be used in the expression
vector (see, e.g., Bitter, et al . , Methods in Enzymology
153 : 516-544 , 1987) . These elements are well known to one of
skill in the art.
In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review see, Current
Protocols in Molecular Biology , Vol. 2, Ed. Ausubel, et al . ,
Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988;
Grant, et al . , "Expression and Secretion Vectors for Yeast," in
Methods in Enzymology , Eds. Wu & Grossman, 1987, Acad. Press,
N.Y., Vol. 153, pp. 516-544, 1987; Glover, DNA Cloning , Vol. II,
IRL Press, Wash., D.C., Ch. 3, 1986; and Bitter, "Heterologous
Gene Expression in Yeast," Methods in Enzymology , Eds. Berger &
Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684, 1987; and The
Molecular Biology of the Yeast Saccharo myces, Eds. Strathern et
al., Cold Spring Harbor Press, Vols. I and II, 1982. A
constitutive yeast promoter such as ADH or LEU2 or an inducible
promoter such as GAL may be used ("Cloning in Yeast," Ch. 3, R.
Rothstein In: DNA Cloning Vol.11, A Practical Approach , Ed. DM
Glover, IRL Press, Wash., D.C., 1986). Alternatively, vectors
may be used which promote integration of foreign DNA sequences
into the yeast chromosome.
An alternative expression system which could be used to express
the proteins of the invention is an insect system. In one such
system, Autographa calif omica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus
grows in Spodoptera frugiperda cells. The sequence encoding a
protein of the invention may be cloned into non-essential
regions (for example, the polyhedrin gene) of the virus and
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placed under control of an AcNPV promoter (for example the
polyhedrin promoter) . Successful insertion of the sequences
coding for a protein of the invention will result in
inactivation of the polyhedrin gene and production of
non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene) . These
recombinant viruses are then used to infect Spodoptera
frugiperda cells in which the inserted gene is expressed, see
Smith, et al . , J*. Viol. 46:584, 1983; Smith, U.S. Patent No.
4, 215, 051.
By "transformation" is meant a permanent or transient genetic
change induced in a cell following incorporation of new DNA
(i.e., DNA exogenous to the cell). Where the cell is a
mammalian cell, a permanent genetic change is generally achieved
by introduction of the DNA into the genome of the cell.
By "transformed cell" or "host cell" is meant a cell (e.g.,
prokaryotic or eukaryotic) into which (or into an ancestor of
which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding a polypeptide of the
invention (i.e., a Methuselah polypeptide), or fragment thereof.
Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to
those skilled in the art. Where the host is prokaryotic, such
as E. coli, competent cells which are capable of DNA uptake can
be prepared from cells harvested after exponential growth phase
and subsequently treated by the CaCl 2 method by procedures well
known in the art. Alternatively, MgCl 2 or RbCl can be used.
Transformation can also be performed after forming a protoplast
of the host cell or by electroporation.
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When the host is a eukaryote, such methods of transfection with
DNA include calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors,
as well as others known in the art, may be used. Eukaryotic
cells can also be cotransf ected with DNA sequences encoding a
polypeptide of the invention, and a second foreign DNA molecule
encoding a selectable phenotype, such as the herpes simplex
thymidine kinase gene. Another method is to use a eukaryotic
viral vector, such as simian virus 40 (SV40) or bovine papilloma
virus, to transiently infect or transform eukaryotic cells and
express the protein. ( Eukaryotic Viral Vectors , Cold Spring
Harbor Laboratory, Gluzman ed. , 1982). Preferably, a eukaryotic
host is utilized as the host cell as described herein. The
eukaryotic cell may be a yeast cell (e.g., Saccharomyces
cerevisiae) , or may be a mammalian cell, including a human cell.
Eukaryotic systems, and mammalian expression systems, allow for
proper post-translational modifications of expressed mammalian
proteins to occur. Eukaryotic cells which possess the cellular
machinery for proper processing of the primary transcript,
glycosylation, phosphorylation, and, advantageously secretion of
the gene product should be used. Such host cell lines may
include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,
Jurkat, HEK-293, and WI38.
Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression may be engineered. For
example, when using adenovirus expression vectors, the nucleic
acid sequences encoding a fusion protein of the invention may be
ligated to an adenovirus transcription/translation control
complex, e.g., the late promoter and tripartite leader sequence.
This chimeric gene may then be inserted in the adenovirus
genome by in vitro or in vivo recombination. Insertion in a
non-essential region of the viral genome (e.g., region El or E3)
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will result in a recombinant virus that is viable and capable of
expressing the Methuselah polypeptide in infected hosts (e.g.,
see Logan & Shenk, Proc . Natl. Acad. Sci. USA, 81:3655-3659,
1984). Alternatively, the vaccinia virus 7.5K promoter may be
used. {e.g., see, Mackett, et al . , Proc. Natl. Acad. Sci. USA,
79:7415-7419, 1982; Mackett, et al . , J. Virol. 49:857-864, 1984;
Panicali, et al., Proc. Natl. Acad. Sci. USA 79:4927-4931,
1982) . Of particular interest are vectors based on bovine
papilloma virus which have the ability to replicate as
extrachromosomal elements (Sarver, et al . , Mol . Cell. Biol.
1:486, 1981) . Shortly after entry of this DNA into mouse cells,
the plasmid replicates to about 100 to 200 copies per cell.
Transcription of the inserted cDNA does not require integration
of the plasmid into the host's chromosome, thereby yielding a
high level of expression. These vectors can be used for stable
expression by including a selectable marker in the plasmid, such
as the neo gene. Alternatively, the retroviral genome can be
modified for use as a vector capable of introducing and
directing the expression of the Methuselah gene in host cells
(Cone Sc Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349-6353,
1984) . High level expression may also be achieved using
inducible promoters, including, but not limited to, the metallo-
thionine IIA promoter and heat shock promoters.
For long-term, high-yield production of recombinant proteins,
stable expression is preferred. Rather than using expression
vectors which contain viral origins of replication, host cells
can be transformed with the cDNA encoding a fusion protein of
the invention controlled by appropriate expression control
elements (e.g., promoter, enhancer, sequences, transcription
terminators, polyadenylation sites, etc.), and a selectable
marker. The selectable marker in the recombinant plasmid
confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form
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Attorney Docket No. 06618/343002
foci which in turn can be cloned and expanded into cell lines.
For example, following the introduction of foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. A
number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., Cell, 11:223, 1977), hypoxanthine -guanine
phosphoribosyltransf erase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA, 48:2026, 1962), and adenine phospho-
ribosyltransf erase (Lowy, et al . , Cell, 22:817, 1980) genes can
be employed in tk~, hgprt" or aprt" cells respectively. Also,
antimetabolite resistance can be used as the basis of selection
for dhfr, which confers resistance to methotrexate (Wigler, et
al., Proc. Natl. Acad. Sci. USA, 77:3567, 1980; O'Hare, et al.,
Proc. Natl. Acad. Sci. USA, 8_:1527, 1981); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA, _78:2072, 1981; neo, which confers resistance to
the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol .
Biol. 150:1, 1981); and hygro, which confers resistance to
hygromycin {Santerre, et al., Gene 3.0:147, 1984) genes.
Recently, additional selectable genes have been described,
namely trpB, which allows cells to utilize indole in place of
tryptophan; hisD, which allows cells to utilize histinol in
place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci.
USA 85:8047, 1988); and ODC (ornithine decarboxylase) which
confers resistance to the ornithine decarboxylase inhibitor,
2- (dif luoromethyl) -DL-ornithine, DFMO (McConlogue L. , In:
Current Communications in Molecular Biology , Cold Spring Harbor
Laboratory, ed., 1987).
The term "primer" as used herein refers to an oligonucleotide,
whether natural or synthetic, which is capable of acting as a
point of initiation of synthesis when placed under conditions in
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which primer extension is initiated or possible. Synthesis of a
primer extension product which is complementary to a nucleic
acid strand is initiated in the presence of nucleoside
triphosphates and a polymerase in an appropriate buffer at a
suitable temperature.
The term "primer" may refer to more than one primer,
particularly in the case where there is some ambiguity in the
information regarding one or both ends of the target region to
be synthesized. For instance, if a nucleic acid sequence is
inferred from a protein sequence, a "primer" generated to
synthesize nucleic acid encoding said protein sequence is
actually a collection of primer oligonucleotides containing
sequences representing all possible codon variations based on
the degeneracy of the genetic code. One or more of the primers
in this collection will be homologous with the end of the target
sequence. Likewise, if a "conserved" region shows significant
levels of polymorphism in a population, mixtures of primers can
be prepared that will amplify adjacent sequences. For example,
primers can be synthesized based upon the amino acid sequence as
set forth in SEQ ID NO: 2 and can be designed based upon the
degeneracy of the genetic code.
The term "restriction endonucleases" and "restriction enzymes"
refers to bacterial enzymes which cut double -stranded DNA at or
near a specific nucleotide sequence.
The term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following
the coding region (leader and trailer) as well as intervening
sequences (introns) between individual coding segments (exons) .
A coding sequence is "operably linked" to another coding
sequence when RNA polymerase will transcribe the two coding
sequences into a single mRNA, which is then translated into a
single polypeptide having amino acids derived from both coding
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Attorney Docket No. 06618/343002
sequences. The coding sequences need not be contiguous to one
another so long as the expressed sequences ultimately process to
produce the desired protein.
A "recombinant" protein or polypeptide refer to proteins or
polypeptides produced by recombinant DNA techniques; i.e.,
produced from cells transformed by an exogenous DNA construct
encoding the desired polypeptide (e.g. a Methuselah polypeptide
of the present invention) . "Synthetic" polypeptides are those
prepared by chemical synthesis.
As used in connection with the present invention the term
"polypeptide" or "protein" refers to a polymer in which the
monomers are amino acid residues which are joined together
through amide bonds. When the amino acids are alpha-amino
acids, either the L-optical isomer or the D-optical isomer can
be used, the L- isomers being preferred. The term "polypeptide"
as used herein is intended to encompass any amino acid sequence
and include modified sequences such as glycoproteins. The term
"polypeptide" is specifically intended to cover naturally
occurring proteins , as well as those which are recombinant ly or
synthetically synthesized, which occur in at least two different
conformations wherein both conformations have the same or
substantially the same amino acid sequence but have different
three dimensional structures. "Fragments" are a portion of a
naturally occurring protein. Fragments can have the same or
substantially the same amino acid sequence as the naturally
occurring protein, "Substantially the same" means that an amino
acid sequence is largely, but not entirely, the same, but
retains a functional activity of the sequence to which it is
related. In general, two amino acid sequences are
"substantially the same" or "substantially homologous" if they
are at least 85% identical. The term "conservative variation" as
used herein denotes the replacement of an amino acid residue by
another, biologically similar residue. Examples of conservative
variations include the substitution of one hydrophobic residue
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Attorney Docket No. 06618/343002
such as isoleucine, valine, leucine or methionine for another,
or the substitution of one polar residue for another, such as
the substitution of arginine for lysine, glutamic for aspartic
acids, or glutamine for asparagine, and the like. The term
"conservative variation" also includes the use of a substituted
amino acid in place of an unsubstituted parent amino acid
provided that antibodies raised to the substituted polypeptide
also immunoreact with the unsubstituted polypeptide. Examples
of conservative substitutions involve amino acids that have the
same or similar properties. Illustrative amino acid
conservative substitutions include the changes of: alanine to
serine; arginine to lysine; asparagine to glutamine or
histidine; aspartate to glutamate; cysteine to serine; glutamine
to asparagine; glutamate to aspartate; glycine to proline;
histidine to asparagine or glutamine; isoleucine to leucine or
valine; leucine to valine or isoleucine; lysine to arginine,
glutamine, or glutamate; methionine to leucine or isoleucine;
phenylalanine to tyrosine, leucine or methionine; serine to
threonine; threonine to serine; tryptophan to tyrosine; tyrosine
to tryptophan or phenylalanine; valine to isoleucine to leucine.
Modifications and substitutions are not limited to replacement
of amino acids. For a variety of purposes, such as increased
stability, solubility, or configuration concerns, one skilled in
the art will recognize the need to introduce, (by deletion,
replacement, or addition) other modifications. Examples of such
other modifications include incorporation of rare amino acids,
dextra-amino acids, glycosylation sites, cytosine for specific
disulfide bridge formation, for example of possible
modifications. The modified peptides can be chemically
synthesized, or the isolated gene can be site-directed
mutagenized, or a synthetic gene can be synthesized and
expressed in bacteria, yeast, baculovirus, tissue culture and so
on.
Attorney Docket No. 06618/343002
A DNA "coding sequence of" or a "nucleotide sequence encoding" a
particular protein, is a DNA sequence which is transcribed and
translated into an protein when placed under the control of
appropriate regulatory sequences.
MTH nucleic acid, polypeptides and method of expression
In one embodiment, the invention provides an isolated
polynucleotide sequence encoding MTH polypeptide. An exemplary
MTH polypeptide of the invention has an amino acid sequence as
set forth in SEQ ID NO: 2. Polynucleotide sequences of the
invention include DNA, cDNA and RNA sequences which encode MTH.
It is understood that all polynucleotides encoding all or a
portion of MTH are also included herein, so long as they encode
a polypeptide with MTH activity (e.g., increased life span or
resistance to stress) . Such polynucleotides include naturally
occurring, synthetic, and intentionally manipulated
polynucleotides. For example, MTH polynucleotide may be
subjected to site-directed mutagenesis. The polynucleotides of
the invention include sequences that are degenerate as a result
of the genetic code. There are 2 0 natural amino acids, most of
which are specified by more than one codon. Therefore, all
degenerate nucleotide sequences are included in the invention as
long as the amino acid sequence of MTH polypeptide encoded by
the nucleotide sequence is functionally unchanged. Also
included are nucleotide sequences which encode MTH polypeptide,
such as SEQ ID N0:1. In addition, the invention also includes
a polynucleotide encoding a polypeptide having the biological
activity of an amino acid sequence of SEQ ID NO: 2 and having at
least one epitope for an antibody immunore active with MTH
polypeptide. However, it is recognized that portions of either
SEQ ID N0:1 or 2 may be excluded to identify fragments of the
polynucleotide sequence or polypeptide sequence. For example,
fragments of SEQ ID NO: 1 or 2 are encompassed by the current
invention, so long as they retain some biological activity
related to mth. A biological activity related to MTH includes
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Attorney Docket No. 06618/343002
for example, antigencity or the ability to affect stress and
life span in an organism.
The polynucleotides of this invention were originally recovered
from Drosophila melanogaster. Thus, the present invention
provides means for isolating the nucleic acid molecules from
other organisms, including humans, encoding the polypeptides of
the present invention. For example, one may probe a gene
library with a natural or artificially designed probe using art
recognized procedures (see, for example: Current Protocols in
Molecular Biology, Ausubel F.M. et al . (EDS.) Green Publishing
Company Assoc. and John Wiley Interscience, New York, 1989,
1992) . It is appreciated by one skilled in the art that probes
can be designed based on the degeneracy of the genetic code to
the sequences set forth in SEQ ID NO: 2.
The invention includes polypeptides having substantially the
same sequence as the amino acid sequence set forth in SEQ ID
NO: 2 or functional fragments thereof, or amino acid sequences
that are substantially identical or the same as SEQ ID NO: 2.
Homology or identity is often measured using sequence analysis
software (e.g. , Sequence Analysis Software Package of the
Genetics Computer Group, University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, WI 53705) . Such
software matches similar sequences by assigning degrees of
homology to various deletions, substitutions and other
modifications. The terms "homology" and "identity" in the context
of two or more nucleic acids or polypeptide sequences, refer to
two or more sequences or subsequences that are the same or have
a specified percentage of amino acid residues or nucleotides
that are the same when compared and aligned for maximum
correspondence over a comparison window or designated region as
measured using any number of sequence comparison algorithms or
by manual alignment and visual inspection.
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Attorney Docket No. 06618/343002
For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference
sequences are entered into a computer, subsequence coordinates
are designated, if necessary, and sequence algorithm program
parameters are designated. Default program parameters can be
used, or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
A "comparison window", as used herein, includes reference to a
segment of any one of the number of contiguous positions
selected from the group consisting of from 20 to 600, usually
about 50 to about 200, more usually about 100 to about 150 in
which a sequence may be compared to a reference sequence of the
same number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequence for
comparison are well-known in the art. Optimal alignment of
sequences for comparison can be conducted, e.g., by the local
homology algorithm of Smith & Waterman, Adv. Appl . Math. 2:482
(1981) , by the homology alignment algorithm of Needleman &
Wunsch, J. Mol. Biol 48:443 (1970), by the search for similarity
method of person & Lipman, Proa. JVatl. Acad. Sci. USA 85.-2444
(1988) , by computerized implementations of these algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Dr. ,
Madison, WI) , or by manual alignment and visual inspection.
On example of a useful algorithm is BLAST and BLAST 2.0
algorithms, which are described in Altschul et al . , Nuc. Acids
Res. 25:3389-3402 (1977) and Altschul et al . , J. Mol. Biol.
215:403-410 (1990), respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Attorney Docket No. 06618/343002
Biotechnology Information (http://www.ncbi.nlm.nih.gov/) . This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in
a database sequence. T is referred to as the neighborhood word
score threshold (Altschul et al . , supra). These initial
neighborhood word hits act as seeds for initiating searches to
find longer HSPs containing them. The word hits are extended in
both directions along each sequence for as far as the cumulative
alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) . For
amino acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction
are halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative- scoring residue alignments; or the end of either
sequence is reached. The BLAST algorithm parameters W, T, and X
determine the sensitivity and speed of the alignment. The
BLASTN program (for nucleotide sequences) uses as defaults a
wordlength (W) or 11, an expectation (E) or 10, M=5, N=-4 and a
comparison of both strands. For amino acid sequences, the
BLASTP program uses as defaults a wordlength of 3, and
expectations (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))
alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Natl. Acad. Sci. USA 90:5873 (1993)). One measure of
similarity provided by BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the
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Attorney Docket No. 06618/343002
probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic
acid is considered similar to a references sequence if the
smallest sum probability in a comparison of the test nucleic
acid to the reference nucleic acid is less than about 0.2, more
preferably less than about 0.01, and most preferably less than
about 0.001.
A "substantially pure polypeptide" is an MTH polypeptide which
has been separated from components which naturally accompany it.
Typically, the polypeptide is substantially pure when it is at
least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the preparation is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, MTH polypeptide. A substantially pure MTH polypeptide
may be obtained, for example, by extraction from a natural
source (e.g., an insect cell); by expression of a recombinant
nucleic acid encoding an MTH polypeptide; or by chemically
synthesizing the protein. Purity can be measured by any
appropriate method, e.g., by column chromatography,
polyacrylamide gel electrophoresis, or by HPLC analysis.
MTH polypeptides of the present invention include peptides, or
full length protein, that contains substitutions, deletions, or
insertions into the protein backbone, that would still leave an
approximately 50%-70% homology to the original protein over the
corresponding portion. A yet greater degree of departure from
homology is allowed if like-amino acids, i.e. conservative amino
acid substitutions, do not count as a change in the sequence.
In addition to polypeptides of the invention, specifically
disclosed herein is a DNA sequence for MTH represented by SEQ ID
NO-.l, DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridiza-
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Attorney Docket No. 06618/343002
tion or computer-based techniques which are well known in the
art. These include, but are not limited to: 1) hybridization of
genomic libraries with probes to detect homologous nucleotide
sequences; 2) antibody screening of expression libraries to
detect cloned DNA fragments with shared structural features; 3)
polymerase chain reaction (PCR) on genomic DNA using primers
capable of annealing to the DNA sequence of interest; and 4}
computer searches of sequence databases for similar sequences.
The polynucleotide encoding MTH includes the nucleotide sequence
in FIGURE 3 (SEQ ID N0:1), as well as nucleic acid sequences
complementary to that sequence. When the sequence is RNA, the
deoxyribonucleotides A, G, C, and T of SEQ ID NO:l are replaced
by ribonucleotides A, G, C, and U, respectively. Also included
in the invention are fragments (portions) of the above -described
nucleic acid sequences that are at least 15 bases in length,
which is sufficient to permit the fragment to selectively
hybridize to DNA that encodes the protein of FIGURE 3 {e.g., SEQ
ID NO: 2) . "Selective hybridization" as used herein refers to
hybridization under moderately stringent or highly stringent
physiological conditions (See, for example, the techniques
described in Maniatis et a2 . , 1989 Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.,
incorporated herein by reference) , which distinguishes related
from unrelated nucleotide sequences.
In nucleic acid hybridization reactions, the conditions used to
achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example,
the length, degree of complementarity, nucleotide sequence
composition (e.g., GC v. AT content), and nucleic acid type
(e.gr., RNA v. DNA) of the hybridizing regions of the nucleic
acids can be considered in selecting hybridization conditions.
An additional consideration is whether one of the nucleic acids
is immobilized, for example, on a filter.
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Attorney Docket No. 06618/343002
An example of progressively higher stringency conditions is as
follows: 2 x SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2 x SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at
about 42°C (moderate stringency conditions); and 0.1 x SSC at
about 68°C (high stringency conditions) . Washing can be carried
out using only one of these conditions, e.g., high stringency
conditions, or each of the conditions can be used, e.g., for 10-
15 minutes each, in the order listed above, repeating any or all
of the steps listed. However, as mentioned above, optimal
conditions will vary, depending on the particular hybridization
reaction involved, and can be determined empirically.
Oligonucleotides encompassed by the present invention are also
useful as primers for nucleic acid amplification reactions. In
general, the primers used according to the method of the
invention embrace oligonucleotides of sufficient length and
appropriate sequence which provides specific initiation of
polymerization of a significant number of nucleic acid molecules
containing the target nucleic acid under the conditions of
stringency for the reaction utilizing the primers. In this
manner, it is possible to selectively amplify the specific
target nucleic acid sequence containing the nucleic acid of
interest. Specifically, the term "primer" as used herein refers
to a sequence comprising two or more deoxyribonucleotides or
ribonucleotides, preferably at least eight, which sequence is
capable of initiating synthesis of a primer extension product
that is substantially complementary to a target nucleic acid
strand. The oligonucleotide primer typically contains 15-22 or
more nucleotides, although it may contain fewer nucleotides as
long as the primer is of sufficient specificity to allow
essentially only the amplification of the specifically desired
target nucleotide sequence (i.e., the primer is substantially
complementary) .
Attorney Docket No. 06618/343002
Amplified products may be detected by Southern blot analysis,
without using radioactive probes. In such a process, for
example, a small sample of DNA containing a very low level of
MTH nucleotide sequence is amplified and analyzed via a Southern
blotting technique known to those of skill in the art. The use
of non-radioactive probes or labels is facilitated by the high
level of the amplified signal.
MTH polynucleotide of the invention is derived from an insect
(e.g., Drosophila) . Screening procedures which rely on nucleic
acid hybridization make it possible to isolate any gene sequence
from any organism, provided the appropriate probe is available.
% For example, it is envisioned that such probes can be used to
CI identify other homologs of the tilth family of factors in insects
^1 or, alternatively, in other organisms such as mammals, e.g.,
humans. In accomplishing this, oligonucleotide probes, which
correspond to a part of the sequence encoding the protein in
s question, can be synthesized chemically. This requires that
il short, oligopeptide stretches of amino acid sequence must be
known. The DNA sequence encoding the protein can be deduced
from the genetic code, however, the degeneracy of the code must
J7 be taken into account. It is possible to perform a mixed
addition reaction when the sequence is degenerate. This
includes a heterogeneous mixture of denatured double -stranded
DNA. For such screening, hybridization is preferably performed
on either single-stranded DNA or denatured double -stranded DNA.
Hybridization is particularly useful in the detection of DNA
clones derived from sources where an extremely low amount of
mRNA sequences relating to the polypeptide of interest are
present. In other words, by using stringent' hybridization
conditions directed to avoid non-specific binding, it is
possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of
the target DNA to that single probe in the mixture which is its
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Attorney Docket No. 06618/343002
complete complement (Wallace, et al . , Nucl . Acid Res., .9:879,
1981) .
When the entire sequence of amino acid residues of the desired
polypeptide is not known, the direct synthesis of DNA sequences
is not possible and the method of choice is the synthesis of
cDNA sequences. Among the standard procedures for isolating
cDNA sequences of interest is the formation of plasmid- or
phage -carrying cDNA libraries which are derived from reverse
transcription of mRNA which is abundant in donor cells that have
a high level of genetic expression. When used in combination
with polymerase chain reaction technology, even rare expression
products can be cloned,
DNA sequences encoding MTH can be expressed in vitro by DNA
transfer into a suitable host cell. "Host cells" are cells in
which a vector can be propagated and its DNA expressed. The
term also includes any progeny of the subject host cell. It is
understood that all progeny may not be identical to the parental
cell since there may be mutations that occur during replication.
However, such progeny are included when the term "host cell" is
used.
In the present invention, the MTH polynucleotide sequences may
be inserted into a recombinant expression vector. The term
"recombinant expression vector" refers to a plasmid, virus or
other vehicle known in the art that has been manipulated by
insertion or incorporation of the MTH genetic sequences. Such
expression vectors contain a promoter sequence which facilitates
the efficient transcription of the inserted genetic sequence of
the host. The expression vector typically contains an origin of
replication, a promoter, as well as specific genes which allow
phenotypic selection of the transformed cells. Vectors suitable
for use in the present invention include those described above.
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Attorney Docket No. 06618/343002
Polynucleotide sequences encoding MTH can be expressed in either
prokaryotes or eukaryotes. Hosts can include microbial, yeast,
insect and mammalian organisms. Such vectors are used to
incorporate DNA sequences of the invention.
Methods which are well known to those skilled in the art can be
used to construct expression vectors containing the MTH coding
sequence and appropriate transcriptional/translational control
signals. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo
recombination/genetic techniques. (See, for example, the
techniques described in Maniatis et al., 1989, Molecular Cloning
A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.)
The genetic construct can be designed to provide additional
benefits, such as, for example addition of C- terminal or N-
terminal amino acid residues that would facilitate purification
by trapping on columns or by use of antibodies. All those
methodologies are cumulative. For example, a synthetic gene can
later be mutagenized. The choice as to the method of producing
a particular construct can easily be made by one skilled in the
art based on practical considerations: size of the desired
peptide, availability and cost of starting materials, etc. All
the technologies involved are well established and well known in
the art. See, for example, Ausubel et al . , Current Protocols in
Molecular Biology, Volumes 1 and 2 (1987) , with supplements, and
Maniatis et al . , Molecular Cloning, a Laboratory Manual, Cold
Spring Harbor Laboratory (1989) . Yet other technical references
are known and easily accessible to one skilled in the art.
Antibodies that bind to MTH
In another embodiment, the present invention provides antibodies
that bind to MTH. Such antibodies are useful for research and
diagnostic tools in the study of biological stress and life
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Attorney Docket No. 06618/343002
span, and associated pathologies in general. Such antibodies
may be administered alone or contained in a pharmaceutical
composition comprising antibodies against MTH and other reagents
effective as modulators of biological stress and life span both
in vitro and in vivo.
The term "epitope", as used herein, refers to an antigenic
determinant on an antigen, such as a MTH polypeptide, to which
the paratope of an antibody, such as an MTH-specific antibody,
binds. Antigenic determinants usually consist of chemically
active surface groupings of molecules, such as amino acids or
sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics .
Antibodies which bind to the MTH polypeptide of the invention
can be prepared using an intact polypeptide or fragments
containing small peptides of interest as the immunizing antigen.
The polypeptide or a peptide used to immunize an animal can be
derived from translated cDNA or chemical synthesis which can be
conjugated to a carrier protein, if desired. Such commonly used
carriers which are chemically coupled to the peptide include
keyhole limpet hemocyanin (KLH) , thyroglobulin, bovine serum
albumin (BSA) , and tetanus toxoid. The coupled peptide is then
used to immunize the animal (e.g., a mouse, a rat, or a rabbit) .
If desired, polyclonal or monoclonal antibodies can be further
purified, for example, by binding to and elution from a matrix
to which the polypeptide or a peptide to which the antibodies
were raised is bound. Those of skill in the art will know of
various techniques common in the immunology arts for
purification and/or concentration of polyclonal antibodies, as
well as monoclonal antibodies (See for example, Coligan, et al. r
Unit 9, Current Protocols in Immunology, Wiley Interscience,
1991, incorporated by reference) .
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Attorney Docket No. 06618/343002
It is also possible to use the anti-idiotype technology to
produce monoclonal antibodies which mimic an epitope. For
example, an anti- idiotypic monoclonal antibody made to a first
monoclonal antibody will have a binding domain in the
hypervariable region which is the "image" of the epitope bound
by the first monoclonal antibody.
An antibody suitable for binding to MTH is specific for at least
one portion of an extracellular region of the MTH polypeptide,
as shown in Figure 3 {SEQ ID NO: 2) . For example, one of skill
in the art can use the peptides to generate appropriate
antibodies of the invention. Antibodies .of the invention
include polyclonal antibodies, monoclonal antibodies, and
fragments of polyclonal and monoclonal antibodies.
The preparation of polyclonal antibodies is well-known to those
skilled in the art. See, for example, Green et al . , Production
of Polyclonal Antisera, in Immunochemical Protocols (Manson,
ed.), pages 1-5 (Humana Press 1992); Coligan et al . , Production
of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters , in
Current Protocols in Immunology, section 2.4.1 (1992), which are
hereby incorporated by reference .
The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature,
256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow
et al . , Antibodies; A Laboratory Manual , page 726 (Cold Spring
Harbor Pub. 1988) , which are hereby incorporated by reference.
Briefly, monoclonal antibodies can be obtained by injecting mice
with a composition comprising an antigen, verifying the presence
of antibody production by removing a serum sample, removing the
spleen to obtain B lymphocytes, fusing the B lymphocytes with
myeloma cells to produce hybridomas, cloning the hybridomas,
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Attorney Docket No. 06618/343002
selecting positive clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures. Monoclonal antibodies can be isolated and purified
from hybridoma cultures by a variety of well-established
techniques. Such isolation techniques include affinity
chromatography with Protein-A Sepharose, size-exclusion
chromatography, and ion-exchange chromatography. See, e.g.,
Coligan et al . , sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;
Barnes et al . , Purification of Immunoglobulin G (IgG) , in
Methods in Molecular Biology, Vol. 10, pages 79-104 (Humana
Press 1992) . Methods of in vitro and in vivo multiplication of
monoclonal antibodies is well-known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 164 0
medium, optionally replenished by a mammalian serum such as
fetal calf serum or trace elements and growth- sustaining
supplements such as normal mouse peritoneal exudate cells,
spleen cells, bone marrow macrophages. Production in vitro
provides relatively pure antibody preparations and allows scale-
up to yield large amounts of the desired antibodies. Large
scale hybridoma cultivation can be carried out by homogenous
suspension culture in an airlift reactor, in a continuous
stirrer reactor, or in immobilized or entrapped cell culture.
Multiplication in vivo may be carried out by injecting cell
clones into mammals histocompatible with the parent cells, e.g.,
osyngeneic mice, to cause growth of antibody-producing tumors.
Optionally, the animals are primed with a hydrocarbon,
especially oils such as pristane (tetramethylpentadecane) prior
to injection. After one to three weeks, the desired monoclonal
antibody is recovered from the body fluid of the animal.
Therapeutic applications for antibodies disclosed herein are
also part of the present invention. For example, antibodies of
the present invention may also be derived from subhuman primate
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Attorney Docket No. 06618/343002
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in Goldenberg
et al. f International Patent Publication WO 91/11465 (1991) and
Losman et al , , Int. J. Cancer, 46:310 (1990), which are hereby
incorporated by reference.
Alternatively, a therapeutically useful anti-MTH antibody may be
derived from a "humanized" monoclonal antibody. Humanized
monoclonal antibodies are produced by transferring mouse
complementarity determining regions from heavy and light
variable chains of the mouse immunoglobulin into a human
variable domain, and then substituting human residues in the
framework regions of the murine counterparts. The use of
antibody components derived from humanized monoclonal antibodies
obviates potential problems associated with the immunogenicity
of murine constant regions. General techniques for cloning
murine immunoglobulin variable domains are described, for
example, by Orlandi et al . , Proc. Nat'l Acad. Sci. USA, 86:3833
(1989) , which is hereby incorporated in its entirety by
reference. Techniques for producing humanized monoclonal
antibodies are described, for example, by Jones et al., Nature,
321: 522 (1986); Riechmann et al . , Nature, 332: 323 (1988);
Verhoeyen et al . , Science, 239 :1534 (1988); Carter et al . ,
Proc. Nat'l Acad. Sci. USA, 89:4285 (1992); Sandhu, Crit. Rev.
Biotech., 12:437 (1992); and Singer et al . , J. Immunol.,
150:2844 (1993), which are hereby incorporated by reference.
Antibodies of the invention also may be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library. See, for example, Barbas et al., Methods: A Companion
to Methods in Enzymology, Vol. 2, page 119 (1991); Winter et
al., Ann. Rev. Immunol. 12: 433 (1994), which are hereby
incorporated by reference. Cloning and expression vectors that
are useful for producing a human immunoglobulin phage library
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Attorney Docket No. 06618/343002
can be obtained, for example, from STRATAGENE Cloning Systems
(La Jolla, CA) .
In addition, antibodies of the present invention may be derived
from a human monoclonal antibody. Such antibodies are obtained
from transgenic mice that have been "engineered" to produce
specific human antibodies in response to antigenic challenge.
In this technique, elements of the human heavy and light chain
loci are introduced into strains of mice derived from embryonic
stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody- secreting hybridomas.
CI Methods for obtaining human antibodies from transgenic mice are
Jfl described by Green et al . , Nature Genet., 7:13 (1994); Lonberg
,2 et al., Nature, 368 :856 (1994); and Taylor et al . , Int.
Immunol . , 6.: 579 (1994) , which are hereby incorporated by
m reference.
■sr. *
^ Antibody fragments of the present invention can be prepared by
proteolytic hydrolysis of the antibody or by expression in E.
q coli of DNA encoding the fragment. Antibody fragments can be
f?;: obtained by pepsin or papain digestion of whole antibodies by
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to
provide a 5S fragment denoted F(ab') 2 . This fragment can be
further cleaved using a thiol reducing agent, and optionally a
blocking group for the sulfhydryl groups resulting from cleavage
of disulfide linkages, to produce 3.5S Fab' monovalent
fragments. Alternatively, an enzymatic cleavage using pepsin
produces two monovalent Fab' fragments and an Fc fragment
directly. These methods are described, for example, by
Goldenberg, U.S. patents No. 4,036,945 and No. 4,331,647, and
references contained therein. These patents are hereby
incorporated in their entireties by reference. .See also
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Attorney Docket No. 06618/343002
Nisonhoff et al., Arch. Biochem. Biophys, . 89:230 (1960);
Porter, Biochem. J. , 73:119 (1959); Edelman et al., Methods in
Enzymology, Vol. 1, page 422 (Academic Press 1967); and Coligan
et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light -heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments
bind to the antigen that is recognized by the intact antibody.
For example, Fv fragments comprise an association of V H and V L
chains. This association may be noncovalent, as described in
Inbar et al . , Proc. Nat'l Acad. Sci. USA, 69:2659 (1972).
Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross -linked by chemicals such
as glutaraldehyde . See, e.g., Sandhu, supra. Preferably, the
Fv fragments comprise V H and V L chains con-
nected by a peptide linker. These single-chain antigen binding
proteins (sFv) are prepared by constructing a structural gene
comprising DNA sequences encoding the V H and V L domains connected
by an oligonucleotide. The structural gene is inserted into an
expression vector, which is subsequently introduced into a host
cell such as E. coll. The recombinant host cells synthesize a
single polypeptide chain with a linker peptide bridging the two
V domains. Methods for producing sFvs are described, for
example, by Whitlow et al . , Methods: A Companion to Methods in
Enzymology, Vol. 2, page 97 (1991); Bird et al., Science,
242:423 (1988); Ladner et al . , U.S. patent No. 4,946,778; Pack
et al., Bio /Technology , 11:1271 (1993); and Sandhu, supra.
Another form of an antibody fragment is a peptide coding for a
single complementarity- determining region (CDR) . CDR peptides
("minimal recognition units") can be obtained by constructing
Attorney Docket No. 06618/343002
genes encoding the CDR of an antibody of interest. Such genes
are prepared, for example, by using the polymerase chain
reaction to synthesize the variable region from RNA of
antibody-producing cells. See, for example, Larrick et al.,
Methods: A Companion to Methods in Enzymology, Vol. 2, page 106
(1991) .
When used for immunotherapy, the monoclonal antibodies,
fragments thereof, or both, of the invention that bind to mth
may be unlabeled or labeled with a therapeutic agent. These
agents can be coupled either directly or indirectly to the
monoclonal antibodies of the invention. One example of indirect
coupling is by use of a spacer moiety. These spacer moieties,
in turn, can be either insoluble or soluble (Diener, et al . ,
Science, 231 : 148 , 1986) and can be selected to enable drug
release from the monoclonal antibody molecule at the target
site. Examples of therapeutic agents which can be coupled to
the monoclonal antibodies of the invention for immunotherapy are
drugs, radioisotopes, lectins, and toxins.
The labeled or unlabeled monoclonal antibodies of the invention
can also be used in combination with therapeutic agents such as
those described above. Especially preferred are therapeutic
combinations comprising the monoclonal antibody of the invention
and immunomodulators and other biological response modifiers.
The dosage ranges for the administration of monoclonal
antibodies of the invention are those large enough to produce
the desired effect (e.g., a change in susceptibility to stress
or life span) . The dosage should not be so large as to cause
adverse side effects, such as unwanted cross-reactions, anaphy-
lactic reactions, and the like. Generally, the dosage will vary
with the age, condition, sex and extent of an mth-associated
disorder or the desired change in the subject and can be
determined by one of skill in the art. The dosage can be
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Attorney Docket No. 06618/343002
adjusted by the individual physician in the event of any
complication* Dosage can vary from about 0.1 mg/kg to about
2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in
one or more dose administrations daily, for one or several days.
Generally, when the monoclonal antibodies of the invention are
administered conjugated with therapeutic agents, lower dosages,
comparable to those used for in vivo diagnostic imaging, can be
used.
The monoclonal antibodies of the invention can be administered
parenterally by injection or by gradual perfusion over time.
The monoclonal antibodies of the invention can be administered
intravenously, intraperitoneally, intramuscularly,
subcutaneously, intracavity, or transdermal ly, alone or in
combination with effector cells.
Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions
or suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's intravenous
vehicles include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the
like. Preservatives and other additives may also be present
such as, for example, antimicrobials, ant i- oxidants, chelating
agents and inert gases and the like.
Modulation of Biological Stress or Life Span
In one embodiment, the invention provides a method for
modulating (e.g., reducing) the effect of biological stress in a
cell or a subject by administering to the cell or subject a
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Attorney Docket No. 06618/343002
therapeutically effective amount of a composition which contains
an MTH polypeptide, or biologically functional fragment thereof
or an agent (e.g, an antibody, ribozyme, antisense molecule, or
double -stranded interf erring RNA molecules) . The term
"biologically functional fragment" encompasses any segment of a
MTH polypeptide that retains the ability to modulate (e.g.,
increase or decrease) biological stress and/or life span.
As used herein, a "therapeutically effective amount" of a
composition containing mth or an mth -modulating agent is defined
as that amount that is effective in modulating a cell's reaction
to a biologic stress and/or modulating life span.
In another embodiment, the present invention provides a method
for modulating mth gene expression and well as methods for
screening for agents which modulate mth gene expression. A cell
or subject is contacted with an agent suspected or known to have
mth gene expression modulating activity. The change in mth gene
expression is then measured as compared to a control or standard
sample. The control or standard sample can be the baseline
expression of the cell or subject prior to contact with the
agent. An agent which modulates mth gene expression may be a
polynucleotide for example. The polynucleotide may be an
antisense, a triplex agent, a ribozyme, or a double -stranded
interf erring RNA. For example, an antisense may be directed to
the structural gene region or to the promoter region of mth.
The agent may be an agonist, antagonist, peptide,
peptidomimetic, antibody, or chemical.
Double -stranded interferring RNA molecules are especially useful
in the present invention. Such molecules act to inhibit
expression of a target gene. For example, double -stranded RNA
molecules can be injected into a target cell or organism to
inhibit expression of a gene and the resultant gene products
activity. It has been found that such double-stranded RNA
molecules are more effective at inhibiting expression than
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Attorney Docket No. 06618/343002
either RNA strand alone. (Fire et al . , Nature, 1998,
19:391 (6669) :806-ll) .
When a disorder is associated with abnormal expression of mth, a
therapeutic approach which directly interferes with the
translation of mth messages into protein is possible.
Alternatively, similar methodology may be used to study mth gene
activity. For example, antisense nucleic acid, double -stranded
interferring RNA or ribozymes could be used to bind to the mth
mRNA or to cleave it. Antisense RNA or DNA molecules bind
specifically with a targeted gene's RNA message, interrupting the
expression of that gene's protein product. The antisense binds
to the messenger RNA forming a double stranded molecule which
cannot be translated by the cell. Antisense oligonucleotides of
about 15-25 nucleotides are preferred since they are easily
synthesized and have an inhibitory effect just like antisense
RNA molecules. In addition, chemically reactive groups, such as
iron-linked ethylenediaminetetraacetic acid (EDTA-Fe) can be
attached to an antisense oligonucleotide, causing cleavage of
the RNA at the site of hybridization. These and other uses of
antisense methods to inhibit the in vitro translation of genes
are well known in the art ( Marcus -Sakur a, Anal. Biochem. ,
172:289, 1988) .
Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, Scientific American, 262:40, 1990) . In the cell,
the antisense nucleic acids hybridize to the corresponding mRNA,
forming a double -stranded molecule. The antisense nucleic acids
interfere with the translation of the mRNA, since the cell will
not translate a mRNA that is double- stranded. Antisense
oligomers of about 15 nucleotides are preferred, since they are
easily synthesized and are less likely to cause problems than
larger molecules when introduced into the target MTH-producing
cell. The use of antisense methods to inhibit the in vitro
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Attorney Docket No. 06618/343002
translation of genes is well known in the art (Marcus -Sakura,
Anal. Biochem. , 172 :289, 1988).
Use of an oligonucleotide to stall transcription is known as the
triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev., 1:227, 1991;
Helene, Anticancer Drug Design, 6:569, 1991) .
Ribozymes are RNA molecules possessing the ability to specifi-
cally cleave other single -stranded RNA in a manner analogous to
DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs , it is possible to
engineer molecules that recognize specific nucleotide sequences
in an RNA molecule and cleave it (Cech, J.Amer.Med. Assn.,
260 :3030, 1988) . A major advantage of this approach is that,
because they are sequence-specific, only mRNAs with particular
sequences are inactivated.
There are two basic types of ribozymes namely, tetrahymena-type
(Hasselhoff, Nature, 334 :585, 1988) and "hammerhead" -type.
Tetrahymena-type ribozymes recognize sequences which are four
bases in length, while "hammerhead" -type ribozymes recognize
base sequences 11-18 bases in length. The longer the recogni-
tion sequence, the greater the likelihood that the sequence will
occur exclusively in the target mRNA species. Consequently,
hammerhead -type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18 -based
recognition sequences are preferable to shorter recognition
sequences .
These and other uses of antisense methods to inhibit the in vivo
translation of genes are well known in the art (e.g., De
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Attorney Docket No. 06618/343002
Mesmaeker, et al., Curr. Opin. Struct. Biol., 5:343, 1995;
Gewirtz, A.M. , et al . , Proc. Natl. Acad. Sci. U.S.A., 93:3161,
1996b; Stein, C.A., Chem. and Biol . 3:319, 1996).
Delivery of antisense, triplex agents, ribozymes, competitive
inhibitors, double -stranded interf erring RNA and the like can be
achieved using a recombinant expression vector such as a
chimeric virus or a colloidal dispersion system or by injection.
Various viral vectors which can be utilized for gene therapy as
taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian
retrovirus. Examples of retroviral vectors in which a single
foreign gene can be inserted include, but are not limited to:
Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma
virus (HaMuSV) , murine mammary tumor virus (MuMTV) , and Rous
Sarcoma Virus (RSV) . A number of additional retroviral vectors
can incorporate multiple genes. All of these vectors can
transfer or incorporate a gene for a selectable marker so that
transduced cells can be identified and generated. By inserting
a polynucleotide sequence of interest into the viral vector,
along with another gene which encodes the ligand for a receptor
on a specific target cell, for example, the vector is now target
specific. Retroviral vectors can be made target specific by
inserting, for example, a polynucleotide encoding a sugar, a
glycolipid, or a protein. Preferred targeting is accomplished
by using an antibody to target the retroviral vector. Those of
skill in the art will know of, or can readily ascertain without
undue experimentation, specific polynucleotide sequences which
can be inserted into the retroviral genome to allow target
specific delivery of the retroviral vector containing the
antisense polynucleotide.
Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles.
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Attorney Docket No. 06618/343002
This assistance can be provided, for example, by using helper
cell lines that contain plasmids encoding all of the structural
genes of the retrovirus under the control of regulatory
sequences within the LTR. These plasmids are missing a
nucleotide sequence which enables the packaging mechanism to
recognize an RNA transcript for encapsidation. Helper cell
lines which have deletions of the packaging signal include but
are not limited to Y2, PA317 and PA12, for example. These cell
lines produce empty virions, since no genome is packaged. If a
retroviral vector is introduced into such cells in which the
packaging signal is intact, but the structural genes are
replaced by other genes of interest, the vector can be packaged
and vector virion produced.
Alternatively, NIH 3T3 or other tissue culture cells can be
directly transfected with plasmids encoding the retroviral
structural genes gag, pol and env, by conventional calcium
phosphate transf ection. These cells are then transfected with
the vector plasmid containing the genes of interest . The
resulting cells release the retroviral vector into the culture
medium.
Another targeted delivery system for polynucleotides is a
colloidal dispersion system. Colloidal dispersion systems
include macromolecule complexes, nanocapsules, microspheres,
beads, and lipid-based systems including oil-in-water emulsions,
micelles, mixed micelles, and liposomes. The preferred
colloidal system of this invention is a liposome. Liposomes are
artificial membrane vesicles which are useful as delivery
vehicles in vitro and in vivo. It has been shown that large
unilamellar vesicles (LUV) , which range in size from 0.2-4.0 jum
can encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules . RNA, DNA and intact virions
can be encapsulated within the aqueous interior and be delivered
to cells in a biologically active form (Fraley, et al., Trends
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Attorney Docket No. 06618/343002
Biochem. Sci., 6:77, 1981). In addition to mammalian cells,
liposomes have been used for delivery of polynucleotides in
plant, yeast and bacterial cells. In order for a liposome to be
an efficient gene transfer vehicle, the following
characteristics should be present: (1) encapsulation of the
genes of interest at high efficiency while not compromising
their biological activity; (2) preferential and substantial
binding to a target cell in comparison to non-target cells; (3)
delivery of the aqueous contents of the vesicle to the target
cell cytoplasm at high efficiency; and (4) accurate and
effective expression of genetic information (Mannino, et al . ,
Biotechniques , 6:682, 1988) .
The composition of the liposome is usually a combination of
phospholipids, particularly high-phase- transition- temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be
used. The physical characteristics of liposomes depend on pH,
ionic strength, and the presence of divalent cations.
Examples of lipids useful in liposome production include phos-
phatidyl compounds, such as phosphatidylglycerol, phosphatidyl-
choline , phosphat idylserine , phosphatidylethanolamine , sphingo-
lipids, cerebrosides, and gangliosides . Particularly useful are
diacylphosphatidyl-glycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms,
and is saturated. Illustrative phospholipids include egg
phosphatidylcholine , dipalmitoylphosphat idylcholine and
distearoylphosphatidylcholine .
The targeting of liposomes has been classified based on
anatomical and mechanistic factors. Anatomical classification
is based on the level of selectivity, for example, organ-
specific, cell-specific, and organelle-specif ic . Mechanistic
targeting can be distinguished based upon whether it is passive
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Attorney Docket No. 06618/343002
or active. Passive targeting utilizes the natural tendency of
liposomes to distribute to cells of the reticuloendothelial
system (RES) in organs which contain sinusoidal capillaries.
Active targeting, on the other hand, involves alteration of the
liposome by coupling the liposome to a specific ligand such as a
monoclonal antibody, sugar, glycolipid, or protein, or by
changing the composition or size of the liposome in order to
achieve targeting to organs and cell types other than the
naturally occurring sites of localization.
The surface of the targeted delivery system may be modified in a
variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer
of the liposome in order to maintain the targeting ligand in
stable association with the liposomal bilayer. Various linking
groups can be used for joining the lipid chains to the targeting
ligand. In general, the compounds bound to the surface of the
targeted delivery system will be ligands and receptors which
will allow the targeted delivery system to find and "home in" on
the desired cells. A ligand may be any compound of interest
which will bind to another compound, such as a receptor.
The agents useful in the method of the invention can be
administered, for in vivo application, parenterally by injection
or by gradual perfusion over time. Administration may be
intravenously, intraperitoneally , intramuscularly,
subcutaneous ly, intracavity, or transdermally . For in vitro
studies the agents may be added or disolved in an appropriate
biologically acceptable buffer and added to a cell or tissue.
Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous
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Attorney Docket No. 06618/343002
carriers include water, alcoholic/aqueous solutions, emulsions
or suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer 1 s intravenous
vehicles include fluid and nutrient replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the
like. Preservatives and other additives may also be present
such as, for example, antimicrobials, ant i -oxidants, chelating
agents and inert gases and the like.
Pharmaceutical compositions
It is envisioned that methods of the present invention can be
used to treat pathologies associated with stress disorders.
Therefore, the present invention encompasses methods for
ameliorating a disorder associated with MTH, including treating
a subject having the disorder, at the site of the disorder, with
a MTH reactive agent. Generally, the terms "treating",
"treatment" and the like are used herein to mean affecting a
subject, tissue or cell to obtain a desired pharmacologic and/or
physiologic effect. The effect may be prophylactic in terms of
completely or partially preventing a disease or sign or symptom
thereof, and/or may be therapeutic in terms of a partial or
complete cure for an infection or disease and/or adverse effect
attributable to the infection or disease. "Treating" as used
herein covers any treatment of, or prevention of, an infection
or disease in an invertebrate, a vertebrate, a mammal,
particularly a human, and includes:
(a) preventing the disease from occurring in a subject that
may be predisposed to the disease, but has not yet been
diagnosed as having it;
(b) inhibiting the disease, i.e., arresting its
development; or
® relieving or ameliorating the disease, i.e., cause
regression of the disease.
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Attorney Docket No. 06618/343002
However, it should be recognized that the compositions and
methods described herein, can be used to bring about a desired
result (e.g., an increase in life span or decrease in
susceptibility to a biological stress) in the absence of a
disease or disorder.
Thus, the invention includes various pharmaceutical compositions
useful for ameliorating symptoms attributable to a MTH-
associated disorder. The pharmaceutical compositions according
to the invention are prepared by bringing an antibody against
MTH, a polypeptide or peptide derivative of MTH, a MTH mimetic,
or a MTH-binding agent according to the present invention into a
form suitable for administration to a subject using carriers,
excipients and additives or auxiliaries. Frequently used
carriers or auxiliaries include magnesium carbonate, titanium
dioxide, lactose, mannitol and other sugars, talc, milk protein,
gelatin, starch, vitamins, cellulose and its derivatives, animal
and vegetable oils, polyethylene glycols and solvents, such as
sterile water, alcohols, glycerol and polyhydric alcohols.
Intravenous vehicles include fluid and nutrient replenishers .
Preservatives include antimicrobial, ant i- oxidants, chelating
agents and inert gases. Other pharmaceutically acceptable
carriers include aqueous solutions, non- toxic excipients,
including salts, preservatives, buffers and the like, as
described, for instance, in Remington* s Pharmaceutical
Sciences, 15th ed. Easton: Mack Publishing Co., 1405-1412,
1461-1487 (1975) and The National Formulary XIV., 14th ed.
Washington: American Pharmaceutical Association (1975), the
contents of which are hereby incorporated by reference. The pH
and exact concentration of the various components of the
pharmaceutical composition are adjusted according to routine
skills in the art. See Goodman and Oilman's The Phamxiacological
Basis for Therapeutics (7th ed.) .
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Attorney Docket No. 06618/343002
The pharmaceutical compositions are preferably prepared and
administered in dose units. Solid dose units are tablets,
capsules and suppositories. For treatment of a subject,
depending on activity of the compound, manner of administration,
nature and severity of the disorder, age and body weight of the
subject, different daily doses are necessary. Under certain
circumstances, however, higher or lower daily doses may be
appropriate. The administration of the daily dose can be
carried out both by single administration in the form of an
individual dose unit or else several smaller dose units and also
by multiple administration of subdivided doses at specific
intervals .
The pharmaceutical compositions according to the invention may
be administered locally or systemically in a therapeutically
effective dose. By "therapeutically effective dose" is meant
the quantity of a compound according to the invention necessary
to prevent, to cure or at least partially arrest the symptoms of
the disease and its complications. Amounts effective for this
use will, of course, depend on the severity of the disease and
the weight and general state of the subject. Typically, dosages
used in vitro may provide useful guidance in the amounts useful
for in situ administration of the pharmaceutical composition,
and animal models may be used to determine effective dosages for
treatment of particular disorders. Various considerations are
described, e.g., in Langer, Science, 249 : 1527, (1990); Gilman
et al . (eds.) (1990), each of which is herein incorporated by
reference .
In one embodiment, the invention provides a pharmaceutical
composition useful for administering a MTH polypeptide, or
nucleic acid encoding a MTH polypeptide, to a subject in need of
such treatment. "Administering" the pharmaceutical composition
of the present invention may be accomplished by any means known
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Attorney Docket No. 06618/343002
to the skilled artisan. Preferably .a "subject" refers to a
mammal, most preferably a human, but may be any organism.
The MTH protein or antibody can be administered parenterally ,
enterically, by injection, rapid infusion, nasopharyngeal
absorption, dermal absorption, rectally and orally.
Pharmaceutically acceptable carrier preparations for parenteral
administration include sterile or aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable
oils such as olive oil, and injectable organic esters such as
ethyl oleate. Carriers for occlusive dressings can be used to
increase skin permeability and enhance antigen absorption.
Liquid dosage forms for oral administration may generally
comprise a liposome solution containing the liquid dosage form.
Suitable solid or liquid pharmaceutical preparation forms are,
for example, granules, powders, tablets, coated tablets,
(micro) capsules, suppositories, syrups, emulsions, suspensions,
creams, aerosols, drops or injectable solution in ampule form
and also preparations with protracted release of active
compounds, in whose preparation excipients and additives and/or
auxiliaries such as disintegrants, binders, coating agents,
swelling agents, lubricants, flavorings, sweeteners and elixirs
containing inert diluents commonly used in the art, such as
purified water.
Screening assay for compounds that affect MTHs
In another embodiment, the invention provides a method for
identifying a compound which modulates mth expression or
activity including incubating components comprising the compound
and a MTH polypeptide, or a recombinant cell expressing a MTH
polypeptide, under conditions sufficient to allow the components
to interact and determining the affect of the compound on the
expression or activity of the gene or polypeptide, respectively.
The term "affect", as used herein, encompasses any means by
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which mth gene expression or protein activity can be modulated.
Such compounds can include, for example / polypeptides,
peptidomimetics, chemical compounds and biologic agents as
described below.
Incubating includes conditions which allow contact between the
test compound and MTH, a cell expressing MTH or nucleic acid
encoding MTH. Contacting includes in solution and in solid
phase. The test ligand (s) /compound may optionally be a
combinatorial library for screening a plurality of compounds.
Compounds identified in the method of the invention can be
further evaluated, detected, cloned, sequenced, and the like,
either in solution or after binding to a solid support, by any
method usually applied to the detection of a specific DNA
sequence such as PCR, oligomer restriction (Saiki, et al.. Bio/-
Technology, 3: 1008-1012 , 1985), oligonucleotide ligation assays
(OLAs) (Landegren, et al . , Science, 241 :1077, 1988), and the
like. Molecular techniques for DNA analysis have been reviewed
(Landegren, et al,, Science, 242.: 229-237 , 1988).
Thus, the method of the invention includes combinatorial
chemistry methods for identifying chemical compounds that bind
to MTH or affect MTH expression or activity. By providing for
the production of large amounts of a MTH, one can identify
ligands or substrates that bind to, modulate, affect the
expression of, or mimic the action of a MTH. For example, a
polypeptide may have biological activity associated with the
wild- type protein, or may have a loss of function mutation due
to a point mutation in the coding sequence, substitution,
insertion, deletion and scanning mutations.
Areas of investigation are the development of therapeutic
treatments. The screening identifies agents that provide
modulation of MTH function in targeted organisms. Of particular
interest are screening assays for agents that have a low
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toxicity for humans. A wide variety of assays may be used for
this purpose, including labeled in vitro protein-protein binding
assays, protein-DNA binding assays, electrophoretic mobility
shift assays, immunoassays for protein binding, and the like.
The purified protein may also be used for determination of
three-dimensional crystal structure, which can be used for
modeling intermolecular interactions , for example.
The term "agent" as used herein describes any molecule, e.g.
protein or pharmaceutical, with the capability of altering or
mimicking the physiological function or expression of a MTH.
Generally, a plurality of assay mixtures are run in parallel
with different agent concentrations to obtain a differential
response to the various concentrations. Typically, one of these
concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection.
Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less
than about 2,500 daltons . Candidate agents comprise functional
groups necessary for structural interaction with proteins,
particularly hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, preferably at least
two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more
of the above functional groups. Candidate agents are also found
among biomolecules including, but not limited to: peptides,
saccharides, fatty acids, steroids, purines, pyrimidines,
derivatives, structural analogs or combinations thereof.
Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For
example, numerous means are available for random and directed
synthesis of a wide variety of organic compounds and
biomolecules, including expression of randomized
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oligonucleotides and oligopeptides. Alternatively, libraries of
natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced.
Additionally, natural or synthetically produced libraries and
compounds are readily modified through conventional chemical,
physical and biochemical means, and may be used to produce
combinatorial libraries. Known pharmacological agents may be
subjected to directed or random chemical modifications, such as
acylation, alkylation, esterif ication and amidif ication to
produce structural analogs.
Where the screening assay is a binding assay, one or more of the
molecules may be joined to a label, where the label can directly
or indirectly provide a detectable signal. Various labels
include radioisotopes, fluorescers, chemiluminescers, enzymes,
specific binding molecules, particles, e.g. magnetic particles,
and the like. Specific binding molecules include pairs, such as
biotin and streptavidin, digoxin and antidigoxin. For the
specific binding members, the complementary member would
normally be labeled with a molecule that provides for detection,
in accordance with known procedures .
A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins,
e.g. albumin, detergents, etc that are used to facilitate
optimal protein-protein binding and/or reduce non-specific or
background interactions. Reagents that improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors
and anti -microbial agents may be used. The mixture of
components are added in any order that provides for the
requisite binding. Incubations are performed at any suitable
temperature, typically between 4 and 40°C. Incubation periods
are selected for optimum activity, but may also be optimized to
facilitate rapid high- throughput screening. Typically between
0.1 and 1 hours will be sufficient.
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Detection of mth in vivo and in vitro
In a further embodiment, the invention provides a method of
detecting mth or a mth-associated disorder in a subject
including contacting a cell component containing mth with a
reagent which binds to the cell component. The cell component
can be nucleic acid, such as DNA or RNA, or it can be protein.
When the component is nucleic acid, the reagent is a nucleic
acid probe or PCR primer. When the cell component is protein,
the reagent is an antibody probe. The probes are detectably
labeled, for example, with a radioisotope, a fluorescent
compound, a bioluminescent compound, a chemiluminescent
compound, a metal chelator or an enzyme. Those of ordinary
skill in the art will know of other labels suitable for binding
to an antibody or nucleic acid probe, or will be able to
ascertain such, using routine experimentation.
For purposes of the invention, an antibody or nucleic acid probe
specific for mth may be used to detect the presence of MTH
polypeptide (using antibody) or polynucleotide (using nucleic
acid probe) in biological fluids or tissues. Any specimen
containing a detectable amount of MTH antigen or polynucleotide
can be used. For example, specimens of this invention include
blood, urine, cerebrospinal fluid, synovial fluid or any tissue*
Another technique which may also result in greater sensitivity
consists of coupling antibodies to low molecular weight haptens.
These haptens can then be specifically detected by means of a
second reaction. For example, it is common to use such haptens
as biotin, which reacts with avidin, or dinitrophenyl,
pyridoxal, and fluorescein, which can react with specific anti-
hapten antibodies.
Alternatively, MTH polypeptide can be used to detect antibodies
to MTH polypeptide in a specimen. The MTH of the invention is
particularly suited for use in immunoassays in which it can be
utilized in liquid phase or bound to a solid phase carrier. In
Attorney Docket No. 06618/343002
addition, MTH used in these assays can be detectably labeled in
various ways .
Examples of immunoassays which can utilize the MTH of the
invention are competitive and noncompetitive immunoassays in
either a direct or indirect format . Examples of such
immunoassays are the radioimmunoassay (RIA) , the sandwich
(immunometric assay) and the Western blot assay. Detection of
antibodies which bind to the MTH of the invention can be done
utilizing immunoassays which run in either the forward, reverse,
or simultaneous modes, including immunohistochemical assays on
physiological samples. The concentration of MTH which is used
will vary depending on the type of immunoassay and nature of the
detectable label which is used. However, regardless of the type
of immunoassay which is used, the concentration of MTH utilized
can be readily determined by one of ordinary skill in the art
using routine experimentation.
The MTH of the invention can be bound to many different carriers
and used to detect the presence of antibody specifically
reactive with the polypeptide. Examples of well-known carriers
include glass, polystyrene, polyvinyl chloride, polypropylene,
polyethylene, polycarbonate, dextran, nylon, amyloses, natural
and modified celluloses, polyacrylamides, agaroses, and
magnetite. The nature of the carrier can be either soluble or
insoluble for purposes of the invention. Those skilled in the
art will know of other suitable carriers for binding MTH or will
be able to ascertain such, using routine experimentation.
There are many different labels and methods of labeling known to
those of ordinary skill in the art. Examples of the types of
labels which can be used in the present invention include
enzymes, radioisotopes, colloidal metals, fluorescent compounds,
chemiluminescent compounds, and bioluminescent compounds.
For purposes of the invention, the antibody which binds to MTH
of the invention may be present in various biological fluids and
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tissues. Any sample containing a detectable amount of
antibodies to MTH can be used. Typically, a sample is a liquid
such as urine, saliva, cerebrospinal fluid, blood, serum and the
like, or a solid or semi-solid such as tissue, feces and the
like .
The monoclonal antibodies of the invention, directed toward MTH,
are also useful for the in vivo detection of antigen. The
detectably labeled monoclonal antibody is given in a dose which
is diagnostically effective. The term "diagnostically
effective" means that the amount of detectably labeled
monoclonal antibody is administered in sufficient quantity to
enable detection of MTH antigen for which the monoclonal
antibodies are specific.
% 4 The concentration of detectably labeled monoclonal antibody
if*!,*?
which is administered should be sufficient such that the binding
f02 to those cells, body fluid, or tissue having MTH is detectable
compared to the background. Further, it is desirable that the
jU detectably labeled monoclonal antibody be rapidly cleared from
^ the circulatory system in order to give the best target -to-
\m background signal ratio.
H " For in vivo diagnostic imaging, the type of detection instrument
available is a major factor in selecting a given radioisotope.
The radioisotope chosen must have a type of decay which is
detectable for a given type of instrument. Still another
important factor in selecting a radioisotope for in vivo
diagnosis is that the half-life of the radioisotope be long
enough so that it is still detectable at the time of maximum
uptake by the target, but short enough so that deleterious
radiation with respect to the host is minimized* Ideally, a
radioisotope used for in vivo imaging will lack a particle
emission, but produce a large number of photons in the 14 0-250
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Attorney Docket No. 06618/343002
key range, which may be readily detected by conventional gamma
cameras .
For in vivo diagnosis, radioisotopes may be bound to
immunoglobulin either directly or indirectly by using an
intermediate functional group. Intermediate functional groups
which often are used to bind radioisotopes which exist as
metallic ions to immunoglobulins are the bifunctional chelating
agents such as diethylenetriaminepentacetic acid (DTPA) and
ethylenediaminetetraacetic acid (EDTA) and similar molecules.
Typical examples of metallic ions which can be bound to the
monoclonal antibodies of the invention are U1 ln, 97 Ru, 67 Ga, 68 Ga,
72 As, 89 Zr, and 201 T1.
The monoclonal antibodies of the invention can also be labeled
with a paramagnetic isotope for purposes of in vivo diagnosis,
as in magnetic resonance imaging (MRI) or electron spin
resonance (ESR) . In general, any conventional method for
visualizing diagnostic imaging can be utilized. Usually gamma
and positron emitting radioisotopes are used for camera imaging
and paramagnetic isotopes for MRI. Elements which are
particularly useful in such techniques include 157 Gd, 55 Mn, 152 Dy,
52 Cr, and 5e Fe.
The monoclonal antibodies of the invention can be used to
monitor the course of amelioration of a stress or MTH-associated
disorder. Thus, by measuring the increase or decrease of MTH
polypeptide present in various body fluids or tissues, it would
be possible to determine whether a particular therapeutic
regiment aimed at ameliorating the disorder is effective.
In another embodiment, nucleic acid probes can be used to
identify mth nucleic acid from a specimen obtained from a
subject. Examples of specimens from which nucleic acid sequence
encoding mth can be derived include insect, human, swine,
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porcine, feline, canine, equine, murine, cervine, caprine,
lupine, leporidine and bovine species.
Oligonucleotide probes, which correspond to a part of the
sequence encoding the protein in question, can be synthesized
chemically. This requires that short,- oligopeptide stretches of
amino acid sequence must be known. The DNA sequence encoding the
protein can be deduced from the genetic code, however, the
degeneracy of the code must be taken into account . It is
possible to perform a mixed addition reaction when the sequence
is degenerate. This includes a heterogeneous mixture of
denatured double -stranded DNA. For such screening,
hybridization is preferably performed on either single- stranded
DNA or denatured double -stranded DNA. Hybridization is
particularly useful in the detection of cDNA clones derived from
sources where an extremely low amount of mRNA sequences relating
to the polypeptide of interest are present. In other words, by
using stringent hybridization conditions directed to avoid
non-specific binding, it is possible, for example, to allow the
autoradiographic visualization of a specific cDNA clone by the
hybridization of the target DNA to that single probe in the
mixture which is its complete complement (Wallace, et al., Nucl.
Acid Res. 9:879, 1981).
In an embodiment of the invention, purified nucleic acid
fragments containing intervening sequences or oligonucleotide
sequences of 10-50 base pairs are radioactively labeled. The
labeled preparations are used to probe nucleic acid from a
specimen by the Southern hybridization technique. Nucleotide
fragments from a specimen, before or after amplification, are
separated into fragments of different molecular masses by gel
electrophoresis and transferred to filters that bind nucleic
acid. After exposure to the labeled probe, which will hybridize
to nucleotide fragments containing target nucleic acid
sequences, binding of the radioactive probe to target nucleic
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acid fragments is identified by autoradiography (see Genetic
Engineering, 1, ed. Robert Williamson, Academic Press, (1981) ,
72-81) . Alternatively, nucleic acid from the specimen can be
bound directly to filters to which the radioactive probe
selectively attaches by binding nucleic acids having the
sequence of interest. Specific sequences and the degree of
binding is quantitated by directly counting the radioactive
emissions .
Where the target nucleic acid is not amplified, detection using
an appropriate hybridization probe may be performed directly on
the separated nucleic acid* In those instances where the target
nucleic acid is amplified, detection with the appropriate
hybridization probe would be performed after amplification.
For the most part, the probe will be detectably labeled with an
atom or inorganic radical, most commonly using radionuclides,
but also heavy metals can be used. Conveniently, a radioactive
label may be employed. Radioactive labels include 32 P, 125 I, 3 H,
14 C, 111 In, 99ra Tc, or the like. Any radioactive label may be
employed which provides for an adequate signal and has
sufficient half -life. Other labels include ligands, which can
serve as a specific binding pair member for a labeled ligand,
and the like. A wide variety of labels routinely employed in
immunoassays can readily be employed in the present assay. The
choice of the label will be governed by the effect of the label
on the rate of hybridization and binding of the probe to mutant
nucleotide sequence. It will be necessary that the label
provide sufficient sensitivity to detect the amount of mutant
nucleotide sequence available for hybridization. Other
considerations will be ease of synthesis of the probe, readily
available instrumentation, ability to automate, convenience, and
the like.
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The manner in which the label is bound to the probe will vary
depending upon the nature of the label* For a radioactive
label, a wide variety of techniques can be employed. Commonly
employed is nick translation with an a 32 P-dNTP or terminal
phosphate hydrolysis with alkaline phosphatase followed by
labeling with radioactive 32 P employing 32 P-NTP and T4
polynucleotide kinase. Alternatively, nucleotides can be
synthesized where one or more of the elements present are
replaced with a radioactive isotope, e.g., hydrogen with
tritium. If desired, complementary labeled strands can be used
as probes to enhance the concentration of hybridized label.
Where other radionucleotide labels are involved, various linking
groups can be employed. A terminal hydroxyl can be esterified,
with inorganic acids, e.g., 32 P phosphate, or 14 C organic acids,
or else esterified to provide linking groups to the label.
Alternatively, intermediate bases may be substituted with
activatable linking groups that can then be linked to a label.
Enzymes of interest as reporter groups will primarily be
hydrolases, particularly esterases and glycosidases, or
oxidoreductases, particularly peroxidases . Fluorescent
compounds include fluorescein and its derivatives, rhodamine and
its derivatives, dansyl, umbellif erone, and so forth.
Chemiluminescers include luciferin, and 2, 3-dihydrophtha-
lazinediones [e.g., luminol) .
The probe can be employed for hybridizing to a nucleotide
sequence affixed to a water insoluble porous support. Depending
upon the source of the nucleic acid, the manner in which the
nucleic acid is affixed to the support may vary. Those of
ordinary skill in the art know, or can easily ascertain,
different supports that can be used in the method of the
invention.
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The nucleic acid from a specimen can be cloned and then spotted
or spread onto a filter to provide a plurality of individual
portions (plaques) . The filter is an inert porous solid
support, e.g., nitrocellulose. Any cells (or phage) present in
the specimen are treated to liberate their nucleic acid. The
lysing and denaturation of nucleic acid, as well as the
subsequent washings, can be achieved with an appropriate
solution for a sufficient time to lyse the cells and denature
the nucleic acid. For lysing, chemical lysing will conveniently
be employed, as described previously for the lysis buffer.
Other denaturation agents include elevated temperatures, organic
reagents, e.g., alcohols, amides, amines, ureas, phenols and
sulfoxides or certain inorganic ions, e.g., thiocyanate and
perchlorate .
After denaturation, the filter is washed in an aqueous buffered
solution, such as Tris, generally at a pH of about 6 to 8,
usually 7. One or more washings may be involved, conveniently
using the same procedure as employed for the lysing and
denaturation. After the lysing, denaturing, and washes have
been accomplished, the nucleic acid spotted filter is dried at
an elevated temperature, generally from about 50°C to 70°C.
Under this procedure, the nucleic acid is fixed in position and
can be assayed with the probe when convenient.
Pre-hybridization may be accomplished by incubating the filter
with the hybridization solution without the probe at a mildly
elevated temperature for a sufficient time to thoroughly wet the
filter. Various hybridization solutions may be employed,
comprising from about 20% to 60% volume, preferably 30%, of an
inert polar organic solvent. A common hybridization solution
employs about 50% formamide, about 0.5 to 1M sodium chloride,
about 0.05 to 0 . 1M sodium citrate, about 0.05 to 0.2% sodium
dodecylsulfate, and minor amounts of EDTA, ficoll (about 300-500
kDa) , polyvinylpyrrolidone, (about 250-500 kDa) and serum
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Attorney Docket No. 06618/343002
albumin. Also included in the hybridization solution will
generally be from about 0.5 to 5 mg/ml of sonicated denatured
DNA, e.g., calf thymus of salmon sperm; and optionally from
about 0.5 to 2% wt/vol glycine. Other additives may also be
included, such as dextran sulfate of from about 100 to 1,000 kDa
and in an amount of from about 8 to 15 weight percent of the
hybridization solution.
The particular hybridization technique is not essential to the
invention. Other hybridization techniques are described by Gall
and Pardue, (Proc. Natl, Acad. Sci. 63_:378, 1969); and John, et
a J . , {Nature, 223 : 582 , 1969). As improvements are made in
hybridization techniques they can readily be applied in the
method of the invention.
The amount of labeled probe present in the hybridization
solution will vary widely, depending upon the nature of the
label, the amount of the labeled probe that can reasonably bind
to the filter, and the stringency of the hybridization.
Generally, substantial excess over stoichiometric concentrations
of the probe will be employed to enhance the rate of binding of
the probe to the fixed target nucleic acid.
In nucleic acid hybridization reactions, the conditions used to
achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example,
the length, degree of complementarity, nucleotide sequence
compound (e.g., GC v. AT content), and nucleic acid type (e.g.,
RNA v. DNA) of the hybridizing regions of the nucleic acids can
be considered in selecting hybridization conditions. An
additional consideration is whether one of the nucleic acids is
immobilized, for example, on a filter.
After the filter has been contacted with a hybridization
solution at a moderate temperature for a period of time
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Attorney Docket No. 06618/343 002
sufficient to allow hybridization to occur, the filter is then
introduced into a second solution having analogous
concentrations of sodium chloride, sodium citrate and sodium
dodecylsulf ate as provided in the hybridization solution. The
time the filter is maintained in the second solution may vary
from five minutes to three hours or more. The second solution
determines the stringency, dissolving cross duplexes and short
complementary sequences. After rinsing the filter at room
temperature with dilute sodium citrate -sodium chloride solution,
the filter may now be assayed for the presence of duplexes in
accordance with the nature of the label. Where the label is
radioactive, the filter is dried and exposed to X-ray film.
The label may also comprise a fluorescent moiety that can then
be probed with a specific fluorescent antibody. Horseradish
peroxidase enzyme can be conjugated to the antibody to catalyze
a chemiluminescent reaction. Production of light can then be
seen on rapid exposure to film.
Growth promotion of cultured cells by mth
In another embodiment, the invention provides a method for
supplementing a culture system with mth or an mth-modulating
agent (e.g., an antibody, antisense or ribozyme molecule) in
order to promote the production and maintenance of an insect or
mammalian cell or cell line. The medium used in the culture
system is preferably a commonly used liquid tissue culture
medium. The medium can be free of serum and supplemented with
various defined components which allow the insect or mammalian
cell to proliferate. Mth or an mth -modulating agent is useful
for supplementing any culture media well known in the art, such
as Grace's insect cell medium or Dulbecco's minimal essential
media (DMEM) , which contains appropriate amino acids, vitamins,
inorganic salts, a buffering agent, and an energy source.
Purified molecules, which include hormones, growth factors,
transport proteins, trace elements, vitamins, and substratum -
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Attorney Docket No. 06618/343002
modifying factors are added to the media to replace biological
fluids .
Transgenic Organisms
The present invention also contemplates transgenic non-human
organisms, including invertebrates, vertebrates and mammals.
For purposes of the subject invention, these animals are
referred to as "transgenic" when such animal has had a
heterologous DNA sequence, or one or more additional DNA
sequences normally endogenous to the animal (collectively
referred to herein as "transgenes" ) chromosomally integrated
into the germ cells of the animal. The transgenic animal
(including its progeny) will also have the transgene integrated
into the chromosomes of somatic cells.
Various methods to make the transgenic animals of the subject
invention can be employed. Generally speaking, three such
methods may be employed* In one such method, an embryo at the
pronuclear stage (a "one cell embryo") is harvested from a
female and the transgene is microinjected into the embryo, in
which case the transgene will be chromosomally integrated into
both the germ cells and somatic cells of the resulting mature
animal. In another such method, embryonic stem cells are
isolated and the transgene incorporated therein by
electroporation, plasmid transfection or microinjection,
followed by reintroduction of the stem cells into the embryo
where they colonize and contribute to the germ line. Methods
for microinjection of mammalian species is described in United
States Patent No. 4,873,191. In yet another such method,
embryonic cells are infected with a retrovirus containing the
transgene whereby the germ cells of the embryo have the
transgene chromosomally integrated therein. When the animals to
be made transgenic are avian, because avian fertilized ova
generally go through cell division for the first twenty hours in
the oviduct, microinjection into the pronucleus of the
fertilized egg is problematic due to the inaccessibility of the
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Attorney Docket No. 06618/343002
pronucleus. Therefore, of the methods to make transgenic
animals described generally above, retrovirus infection is
preferred for avian species, for example as described in U.S.
5,162,215. If microinjection is to be used with avian species,
however, a recently published procedure by Love et al . ,
(Biotechnology, 12, Jan 1994) can be utilized whereby the embryo
is obtained from a sacrificed hen approximately two and one-half
hours after the laying of the previous laid egg, the transgene
is microinjected into the cytoplasm of the germinal disc and the
embryo is cultured in a host shell until maturity. When the
animals to be made transgenic are bovine or porcine,
microinjection can be hampered by the opacity of the ova thereby
making the nuclei difficult to identify by traditional
differential interference-contrast microscopy. To overcome this
problem, the ova can first be centrifuged to segregate the
pronuclei for better visualization.
The "non-human animals 11 of the invention include, for example,
bovine, porcine, ovine and avian animals (e.g., cow, pig, sheep,
chicken, turkey) . The "transgenic non-human animals" of the
invention are produced by introducing " transgenes" into the
germline of the non-human animal. Embryonal target cells at
various developmental stages can be used to introduce
transgenes. Different methods are used depending on the stage of
development of the embryonal target cell. The zygote is the best
target for micro- injection. The use of zygotes as a target for
gene transfer has a major advantage in that in most cases the
injected DNA will be incorporated into the host gene before the
first cleavage (Brinster et al . , Proc. Natl. Acad. Sci . USA
82::4438-4442, 1985) . As a consequence, all cells of the
transgenic non-human animal will carry the incorporated
transgene. This will in general also be reflected in the
efficient transmission of the transgene to offspring of the
founder since 50% of the germ cells will harbor the transgene.
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Attorney Docket No. 06618/343002
The term "transgenic" is used to describe an animal which
includes exogenous genetic material within all of its cells. A
"transgenic" animal can be produced by cross-breeding two
chimeric animals which include exogenous genetic material within
cells used in reproduction. Twenty- five percent of the
resulting offspring will be transgenic i.e., animals which
include the exogenous genetic material within all of their cells
in both alleles. 50% of the resulting animals will include the
exogenous genetic material within one allele and 25% will
include no exogenous genetic material.
In the microinjection method useful in the practice of the
subject invention, the transgene is digested and purified free
from any vector DNA e.g. by gel electrophoresis. It is
preferred that the transgene include an operatively associated
promoter which interacts with cellular proteins involved in
transcription, ultimately resulting in constitutive expression.
Promoters useful in this regard include those from
cytomegalovirus (CMV) , Moloney leukemia virus (MLV) , and herpes
virus, as well as those from the genes encoding metallothionin,
skeletal actin, P-enolpyruvate carboxylase (PEPCK) ,
phosphoglycerate (PGK) , DHFR, and thymidine kinase. Promoters
for viral long terminal repeats (LTRs) such as Rous Sarcoma
Virus can also be employed. When the animals to be made
transgenic are avian, preferred promoters include those for the
chicken p-globin gene, chicken lysozyme gene, and avian leukosis
virus. Constructs useful in plasmid transfection of embryonic
stem cells will employ additional regulatory elements well known
in the art such as enhancer elements to stimulate transcription,
splice acceptors, termination and polyadenylation signals, and
ribosome binding sites to permit translation.
Retroviral infection can also be used to introduce transgene
into a non-human animal, as described above. The developing
non-human embryo can be cultured in vitro to the blastocyst
Attorney Docket No. 06618/343002
stage. During this time, the blastomeres can be targets for
retro viral infection (Jaenich, R., Proc. Natl. Acad. Sci USA
73:1260-1264, 1976). Efficient infection of the blastomeres is
obtained by enzymatic treatment to remove the zona pellucida
(Hogan, et al . (1986) in Manipulating the Mouse Embryo, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The
viral vector system used to introduce the transgene is typically
a replication-defective retro virus carrying the transgene
(Jahner, et al., Proc. Natl. Acad. Sci. USA 82 : 6927-6931, 1985;
Van der Putten, et al . , Proc. Natl. Acad. Sci USA 82 : 6148-6152 ,
1985) . Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus -producing
cells (Van der Putten, supra; Stewart, et al . , EMBO J.
6:383-388, 1987) . Alternatively, infection can be performed at a
later stage. Virus or virus -producing cells can be injected into
the blastocoele (D. Jahner et al . , Nature 298:623-628, 1982).
Most of the founders will be mosaic for the transgene since
incorporation occurs only in a subset of the cells which formed
the transgenic nonhuman animal. Further, the founder may contain
various retro viral insertions of the transgene at different
positions in the genome which generally will segregate in the
offspring. In addition, it is also possible to introduce
transgenes into the germ line, albeit with low efficiency, by
intrauterine retroviral infection of the midgestation embryo (D.
Jahner et al . , supra).
A third type of target cell for transgene introduction is the
embryonal stem cell (ES) . ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with
embryos (M. J. Evans et al . Nature 292 : 154-156 , 1981; M.O.
Bradley et al. f Nature 309: 255-258, 1984; Gossler, et al . ,
Proc. Natl. Acad. Sci USA 83 : 9065-9069, 1986; and Robertson et
al., Nature 322:445-448, 1986). Transgenes can be efficiently
introduced into the ES cells by DNA transfection or by retro
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virus -mediated transduction. Such transformed ES cells can
thereafter be combined with blastocysts from a nonhuman animal .
The ES cells thereafter colonize the embryo and contribute to
the germ line of the resulting chimeric animal. (For review see
Jaenisch, R. , Science 24 0 : 1468-1474, 1988).
"Transformed" means a cell into which (or into an ancestor of
which) has been introduced, by means of recombinant nucleic acid
techniques, a heterologous nucleic acid molecule. "Heterologous"
refers to a nucleic acid sequence that either originates from
another species or is modified from either its original form or
the form primarily expressed in the cell.
"Transgene" means any piece of DNA which is inserted by artifice
into a cell, and becomes part of the genome of the organism
(i.e., either stably integrated or as a stable extrachromosomal
element) which develops from that cell. Such a transgene may
include a gene which is partly or entirely heterologous (i.e.,
foreign) to the transgenic organism, or may represent a gene
homologous to an endogenous gene of the organism. Included
within this definition is a transgene created by the providing
of an RNA sequence which is transcribed into DNA and then
incorporated into the genome. The transgenes of the invention
include DNA sequences which encode mth, and include mth- sense,
antisense, dominant negative encoding polynucleotides, which may
be expressed in a transgenic non-human animal. The term
"transgenic" as used herein additionally includes any organism
whose genome has been altered by in vitro manipulation of the
early embryo or fertilized egg or by any transgenic technology
to induce a specific gene knockout. The term "gene knockout" as
used herein, refers to the targeted disruption of a gene in vivo
with complete or partial loss of function that has been achieved
by any transgenic technology familiar to those in the art. In
one embodiment, transgenic animals having gene knockouts are
those in which the target gene has been rendered nonfunctional
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by an insertion targeted to the gene to be rendered non-
functional by homologous recombination. As used herein, the
term "transgenic" includes any transgenic technology familiar to
those in the art which can produce an organism carrying an
introduced transgene or one in which an endogenous gene has been
rendered non- functional or "knocked out."
In one embodiment, the transgene comprises DNA antisense to the
coding sequence for MTH. In another embodiment, the transgene
comprises DNA encoding an antibody which is able to bind to MTH.
Where appropriate, DNA sequences that encode proteins having
MTH activity but differ in nucleic acid sequence due to the
degeneracy of the genetic code may also be used herein, as may
truncated forms, allelic variants and interspecies homologues.
The invention also includes animals having heterozygous
mutations in mth or partial inhibition of mth function or
expression. Partial loss of function leads to an increase in
resistance to biological stress, increase in the mass of the
organism and an increase in life span. One of skill in the art
would readily be able to determine if a particular mutation or
if an antisense molecule was able to partially inhibit mth. For
example, in vitro testing may be desirable initially by
comparison with wild-type or untreated mth (e.g., comparison of
northern blots to examine a decrease in expression) .
After an embryo has been microinjected, colonized with
transfected embryonic stem cells or infected with a retrovirus
containing the transgene (except for practice of the subj ect
invention in avian species which is addressed elsewhere herein)
the embryo is implanted into the oviduct of a pseudopregnant
female. The consequent progeny are tested for incorporation of
the transgene by Southern blot analysis of blood samples using
transgene specific probes. PCR is particularly useful in this
regard. Positive progeny (GO) are crossbred to produce
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offspring (Gl) which are analyzed for transgene expression by
Northern blot analysis of tissue samples. To be able to
distinguish expression of like-species transgenes from
expression of the animals endogenous mth gene(s), a marker gene
fragment can be included in the construct in the 3 1 untranslated
region of the transgene and the Northern probe designed to probe
for the marker gene fragment. The serum levels of mth can also
be measured in the transgenic animal to establish appropriate
expression. Expression of the mth transgenes, thereby
decreasing the mth in the tissue and serum levels of the
transgenic animals .
Transgenic organisms of the invention are highly useful in the
production of organisms having increased mass for food stuff.
For example, bovine, porcine and other animals commonly used for
food stuff can be produced using the techniques described above
having one allele of mth "knockout" resulting in a
heterozygosity. Such organism will demonstrate an increase in
mass, for example an increase in fat or muscle mass. In
addition, such organisms are useful in the study of age
dependence on gene expression as well as age dependence on
learning ability. For example, the transgenic or mutant flies
of the inevntion can be used to study the effect on learning
during aging. Due to the increased life span of organisms
heterozygous for mth, the learning ability of the organism will
be affected. Conditioned behavior in such mutant animals can be
studied (see, Quinn et al., Proc. Natl, Acad. Sci. USA, 1974,
71 (3) :708-12) .
Kits for detection of MTH
The materials for use in the method of the invention are ideally
suited for the preparation of a kit. Such a kit may comprise a
carrier means being compartmentalized to receive one or more
container means such as vials, tubes, and the like, each of the
container means comprising one of the separate elements to be
used in the method. For example, one of the container means may
Attorney Docket No. 06618/343002
comprise a MTH binding reagent, such as an antibody or nucleic
acid. A second container may further comprise MTH polypeptide.
The constituents may be present in liquid or lyophilized form,
as desired.
One of the container means may comprise a probe which is or can
be detectably labeled. Such probe may be an antibody or
nucleotide specific for a target protein, or fragments thereof,
or a target nucleic acid, or fragment thereof, respectively,
wherein the target is indicative, or correlates with, the
presence of MTH. For example, oligonucleotide probes of the
present invention can be included in a kit and used for
examining the presence of mth nucleic acid, as well as the
quantitative (relative) degree of binding of the probe for
determining the occurrence of specific strongly binding
(hybridizing) sequences, thus indicating the likelihood for a
subject having a cell growth-associated pathology.
The kit may also contain a container comprising a reporter-
means, such as a biotin-binding protein, such as avidin or
streptavidin, bound to a reporter molecule, such as an
enzymatic, fluorescent, or radionucleotide label to identify the
detectably labeled oligonucleotide probe.
Where the kit utilizes nucleic acid hybridization to detect the
target nucleic acid, the kit may also have containers containing
nucleotide (s) for amplification of the target nucleic acid
sequence. When it is desirable to amplify the target nucleic
acid sequence, such as a mth nucleic acid sequence, this can be
accomplished using oligonucleotide (s) that are primers for
amplification. These oligonucleotide primers are based upon
identification of the flanking regions contiguous with the
target nucleotide sequence.
Attorney Docket No. 06S18/343002
The kit may also include a container containing antibodies which
bind to a target protein, or fragments thereof. Thus, it is
envisioned that antibodies which bind to MTH, or fragments
thereof, can be included in a kit.
Without further elaboration, it is believed that one skilled in
the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples
are to be considered illustrative and thus are not limiting of
the remainder of the disclosure in any way whatsoever.
Examples
Example 1: Generation and Identification of Mutants
A set of P-element insertion lines was generated (E. Bier et
al., Genes Dev. 3, 1273-1287 (1989); Spradling et al . , Science,
218, 341-347 (1982)) and screened for ones that outlived the
parent strain (white 1118 ) . Because flies live for months at
ambient laboratory temperature, the screen was conducted at 29°C
to accelerate the process. Methuselah (rath) , was isolated by
its increase in lifespan. The life extension was confirmed at
the standard temperature of 25°C, at which stocks in the
laboratory are maintained. At that temperature, flies
homozygous for this P-element live, on the average, 35% longer
(Fig. 1) .
Example 2 : Stress Resistance
The ability of mth flies to resist stress was then examined. As
shown in Fig. 2A, mth mutant flies were more resistant to
dietary paraquat, a bipyridinium salt which, upon intake by the
cell, is reduced to paraquat radical, subsequently giving rise
to the original paraquat ion plus superoxide anion (Ashton et
al., Mode of Action of Insecticide, John Wiley Interscience, New
York, 1973) . At a concentration of 20 mM administered by
Attorney Docket No. 0S618/343002
feeding in 5% sucrose solution, paraquat rendered normal males
sluggish by 12 hours; at 48 hours, nearly 90% were dead. In
contrast, mth males were still active at 24 hours, and at 48
hours more than 50% were still alive. Similar observations were
made by Arking et al., Dev. Genet., 12, 362-370 (1991), on a
long-lived strain of Drosophila derived by selection, in which
lifespan extension accompanied increased paraquat resistance.
Transgenic Drosophila carrying extra copies of SOD and catalase,
two primary components of the defense system against reactive
oxygen species, had significant increase of lifespan (Orr et
al., Science, 263, 1128-1120 (1994)). Flies transgenic for the
human SOD1 gene also displayed increased lifespan and paraquat
resistance, the degree of effect correlating with dosage of the
transgene (Parks et al., Nature Genetics, 19, 171-174 (1998)).
Although, Applicants are not under any obligation to explain the
mechanism of action of mth, mth may have a higher capacity for
modulating free-radical activity in the free-radical defense
system.
In the starvation test, mth showed over 50% increase in average
survival time over the parent strain (Fig. 2B) . Females were
significantly more resistant than males, suggesting that their
larger body weight may contribute to resistance. (Service et
al., Physiol. Zool., 58, 380-389 (1985) ) reported that, in a
Drosophila stock selectively bred for postponed senescence,
resistance to starvation and lipid content were higher than the
baseline stock. In C. elegans, the mutant daf-2, which exhibits
marked increase in longevity, had extensive fat accumulation
when grown at 25°C (Kimura et al., Science, 277, 942-946 (1997));
it was suggested that a higher metabolic capacity of the daf-2
worm plays a central role in its longevity.
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The results of exposure to high temperature are shown in Fig.
2C. At 36°C, mth mutant flies survived longer. There was little
difference between flies of different sexes, consistent with the
observation of Service et al., Physiol. Zool . , 58, 380-389
(1985) . Heat shock proteins, a class of molecular chaperones,
are thought to counter stress -induced detrimental effects during
aging (Heydari et al . , Proc. Natl. Acad. Sci. USA, 92, 10408-
10412 (1995) ) . In a transgenic fly which harbored 12 additional
copies of the heat -inducible hsp70 gene, there was a positive
correlation between increased life expectancy and elevated hsp70
protein expression (Tatar et al., Nature, 390, 30 (1997)).
Correspondingly, in the long-lived C. elegans mutants, daf-2 and
age-1, resistance to thermal stress was higher than that in
control animals (Lithgow et al . , J. Gerontol., 49, B270-B276
(1994); Lithgow et al . , Proc. Natl. Acad. Sci. USA, 92, 7540-
7544 (1995) ) . Although the inventors are under no duty to
explain the mechanism of function of mth, the increased thermo-
tolerance of mth may result from higher expression of heat shock
proteins and related molecular chaperones.
Example 3 : Identification of Mutant Sequence
To generate P-element insertion lines, females carrying 8 copies
of P{2acW} on a compound X chromosome (C(l)RM) were crossed with
ry Ki P{ry* D2-3}, which carries the transposase. Female
progeny were then crossed individually with w 1118 . New P-element
insertions on autosomes were identified by red eye color in male
progeny. In any cross which generated progeny having different
degrees of red eye color, suggestive of multiple insertions, we
chose a single female with lighter eye color and back-crossed it
to w 1118 for several generations, until the color was homogenous.
Each insertion was mapped to a chromosome by the use of
balancers, tested for homozygous viability and fertility, and
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established as an independent line. By Southern blots of mth
genomic DNA, probed by the ampicillin resistance gene contained
in the P-element construct used to generate mutant lines. To
generate P-element insertion lines, females carrying 8 copies of
p{lacW} (E. Biers et al . , supra) on a compound X chrmosome
(C(l)RM) were crossed with ry Ki P{ry* D2-3}, which carries the
transposase. Female progeny were then crossed individually with
w 1118 . New P-element insertions on autosomes were identified by
red eye color in male progeny. In any cross which generated
progeny having different degrees of red eye color, suggestive of
multiple insertions, we chose a single female with lighter eye
color and back-crossed it to w 1118 for seveal generations, until
the color was homogenous. Each insertion was mapped to a
chromosome by the use of balancers, tested for homozygous
viability and fertility, and established as an independent line.
It was confirmed that mth carries a single P-element insertion
in the genome. Genetic mapping indicated that it is inserted in
the third chromosome. By crossing mth flies to flies harboring
the specific transposase, a line was generated in which the P-
element was precisely excised from the insertion site (as
determined by PCR; see below) . To excise the P-element, mth
females were crossed with ry kiP{ry* D2-3}. The male jump-
starters were then crossed to w;TM3/TM6. Progeny with white
eyes were made homozygous and lines established. Two alleles
were homozygous lethal prior to the LI larval stage; those lines
were thus maintained over the third chromosome balancers, TM3 or
TM6. Eight lines obtained in this manner had lifespans reverted
to that of the parent strain, indicating that the phenotype in
mth was specifically caused by P-element insertion. The
precise-excision strains were used as controls throughout the
study; they behaved similarly to the parental strain in stress
resistance as well, indicating that the P-element insertion was
responsible for both aspects of the phenotype.
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Attorney Docket No. 06618/343002
Two other lines also isolated had imprecise excisions of the P-
element, resulting in DNA deletion adjacent to the insertion
site. Both of these lines, likely representing null alleles of
the mth gene, displayed embryonic lethality in homozygotes ,
suggesting that the gene also plays an essential role in
development. Flies heterozygous for the P-element over an
imprecise excision allele were more resistant to stress than
those homozygous for the P-element, indicating that the mutation
created by the P-element insertion is a hypomorphic allele.
EXAMPLE 4 : The Methuselah gene
Genomic DNA adjacent to the P-element insertion site in the mth
mutant fly was retrieved by plasmid rescue technique (Hamilton
et al., Methods in cell biology. L.S. B. Golstein eds., Academic
press, Inc., San Diego, 1994, vol. 44, pp. 81-94). Plasmid
rescue from Drosophila genomic DNA was performed according to
Hamilton et al., supra. Pst I and EcoR I digestion were used to
clone upstream and downstream genomic fragments (Fig. 3A) .
Among the 21 upstream and 15 downstream clones, all within each
group had identical restriction fragment patterns and nucleotide
sequences (up to at least 500 bases) flanking each end of the
plasmid rescue vector, confirming that there was only a single
P-element insertion in mth. Analysis of the upstream DNA
sequence by BLAST search (Altschul et al . , J. Mol . Biol., 215,
403-410 (1990)) revealed two homologous sequences (Clones
LD08316 and GM02553) in the EST database of the Berkeley
Drosophila Genome Project (BDGP) . The GM02553 clone, albeit
containing regions with 67% identity to the mth nucleotide
sequence, had 17 gaps in the alignment. In contrast, the 747-
nucleotide partial sequence of LD08316 in the BDGP database
displayed greater than 99% identity to the upstream sequence,
without any gap. The calculated smallest sum probability of the
BLAST search was 1.5e-137, well within the range of identical
sequences .
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Attorney Docket No. 06618/343002
A LD08316 clone was obtained via the BDGP and its full sequence
determined (1948 nucleotides; see Fig. 3B) , finding that its
sequence corresponded with the downstream genomic sequence of
mth except for a small (less than 1%) sequence disparity,
probably due to polymorphism among different melanogaster
strains. The cDNA was then used as a probe to isolate the full-
length mth genomic DNA. Three PI plasmids (DS05332, DS03799,
and DS06692) from the BDPG contained the genomic region of the
mth gene. These PI clones have a common contig. DS00539, which
maps at 61C on the third chromosome (BDGP database) . A
corresponding 7 . 9-kilobase
EcoR I fragment from DS06692 was subcloned into pBluescript
vector and the full-length sequence determined (Fig. 3A) . The
P-element insertion in the third intron of the mth gene, may
reduce the level of gene expression by interfering with RNA
splicing, without eliminating the gene function. By polymerase
chain reaction and sequencing, we confirmed that the precise-
excision strains had restored the normal sequence across the
insertion site.
The mth cDNA encodes a single, uninterrupted open reading frame
(Fig. 3B) . The predicted protein sequence has a leader peptide
plus seven hydrophic regions suggestive of transmembrane (TM)
domains (Fig. 3C) . A gapped BLAST search (Altschul et al . ,
Nucleic Acids Res., 25, 3389-3402 (1997)) of this sequence
showed homology to a variety of GTP-binding regulatory protein
(G-protein) -coupled receptors (Fig. 4A) . G-protein- coupled
receptor was also predicted by the Blocks Search program
(Henikoff Sc Henikoff, Genomics, 19, 97-107 (1994)). The amino
acid residues between TM5 and TM6 , especially those near the
transmembranes, are highly basic, a feature shared by many G-
protein- linked receptors and known, in some cases, to interact
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Attorney Docket No. 06618/343002
directly with G-proteins (Kobilka et al . , Science, 24:0, 1310-
1316 (1988)). Interestingly, homology was found mainly in the
TM regions. The N- terminal segment prior to the first TM domain
was not found to share homology with any known sequence.
Therefore, despite the structural conservation in the TM
regions, the overall homology score with any given sequence was
diminished. The mth gene appears to represent a novel member of
the seven-TM protein superfamily.
G-protein-coupled receptors are involved in a remarkably diverse
array of biological activities, including neurotransmission,
hormone physiology, drug response, morphogenetic
differentiation, embryonic development, and transduction of
stimuli such as light and odorants (Watson & Arkinstall, The G-
protein linked receptor facts book (Academic Press, London,
1994)). The data indicate that mth is a G-protein-coupled
receptor involved in stress response and biological aging. By
regulating an associated G-protein and thus its downstream
pathway, the normal mth gene may maintain homeostasis and
metabolism, playing a central role in modulating molecular
events in response to stress. The embryonic lethality of all
the null alleles demonstrates that at least some activity of the
mth gene is essential for survival. When mutated, the
intermediate level of ^ expression of a hypomorphic allele might
adjust response to stress in a way that is more favorable for
survival, whereas full expression of the normal gene exceeds the
optimum value. The delicate balance among the embryonic
lethality of a null allele, enhance longevity of a hypomorphic
allele, and the normal wild phenotype suggests that the level of
mth gene expression is an important component of the system
controlling lifespan. Investigation of the gene's function and
associated pathways, should lead to better understanding of
mechanisms relevant to aging.
Attorney Docket No. 06618/343002
Since lifespan and stress response are closely related, genetic
screening by stress resistance provides an effective alternative
to the much slower direct screening for lifetime. The ability
of the mth fly to resist various kinds of stress is notable,
since there are likely to exist differences in pathways of
response to individual forms of stress. For instance, in the
process of this study, we have also obtained a Drosphila mutant
line that is resistant to starvation, but not heat.
Example 5: Expression of Methuselah
In order to determine the expression of MTH in wildtype (e.g.,
w 1118 ) flies compared to mth mutant flies, labeled antisense
probes were used. The antisense probes were derived from the
cDNA sequence for mth, as described above, and enzymatically
labeled (although any type of label known in the art can be
used) . Figure 5 shows the comparison of mth expression in w 1118
flies compared to the Methuselah mutants in the head region of
the fly. A reduction of about 10 -fold was seen in expression of
MTH in the mutant flies compared to the controls. Figure 6,
shows similar expression in the thoracic region of the fly.
Figure 7 shows the results of an RNA protection assay using a
labeled antisense RNA to the mth cDNA sequence. The gel
compares the expression of mth in wildtype flies and mth mutant
flies (P+/mth+) . Lane 5 represents RNA from mth mutant adult
flies, demonstrating a reduction in expression of mth in P+/mth+
Drosophila compared to lane 6. Lane 6 shows RNA from wildtype
Drosophila (mth+/mth+) . Lane 7 and 8 demonstrate the difference
in expression of mth in mutant flies (lane 7) compared to
wildtype (lane 8) in Drosophila embryos.
Example 6: Monoclonal Antibodies to MTH
A monoclonal antibody was raised against the MTH sequence
AHRQERKQKLNSDK using techniques well known to those of skill in
the art, as described above.
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Using the monoclonal antibody, above, localization and
expression of mth in Drosophila mth-mutants and wildtype flies
was performed. The antibody was incubated with sections of
Drosophila and developed with anti-mouse secondary antibody and
FITC. FIG. 8 demonstrates the localiation and expression. The
left series of panels represents wildtype flies (i.e., panels A,
C, E, and G) and the right series of panels represents the mth
mutant flies (i.e., panels B, D, F, and H) . Panels A-B are from
the trunk thoracic muscles of the flies. Panels C-D are from
the ventral layer of the thoracic region. Panels E-F are from
leg muscles of the flies. Panels G-H are from the proboscis
muscle. The mth mutant flies have a reduction of MTH protein in
all segments of the Drosophila tested.
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